Issued  October  2, 1912. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

S^  BUREAU  OF  ANIMAL  INDUSTRY.— Bulletin  150. 

—  6 

=  3  A.  D.  MELVIN,  Chief  of  Bureau. 


i  HE  BACTERIOLOGY  OF  CHEDDAR  CHEESE. 

m  OF  CALIFORNIA, 
LLBHAF^Y, 

BY 


E.  G.  HASTINGS, 

Bacteriologist,  Wisconsin  Agricultural  Experiment  Station^ 

ALICE  C.  EVANS, 

Bacteriologist,  Dairy  Division,  Bureau  of  Animal  Industry^ 
AND 

[  E.  B.  HART, 

^S  Chemist,  Wisconsin  Agricultural  Experiment  Station. 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE. 

1912 


Issued  October  2,  1<>12. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— Bulletin  150. 

A.  D.  MELVIN,  Chief  of  Bureau. 


THE  BACTERIOLOGY  OF  CHEDDAR  CHEESE. 


BY 


E.  G.  HASTINGS, 
Bacteriologist,  Wisconsin  Agricultural  Experiment  Station, 

ALICE  C.  EVANS, 

Bacteriologist,  Dairy  Diinsion,  Bureau  of  Animal  Industry , 
AND 

E.  B.  HART, 

Chemist,  Wisconsin  Agricultural  Experiment  Station. 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE, 

1912 


BUREAU  OF  ANIMAL  INDUSTRY. 


Chief:  A.  D.  Mbxvin.  , 

Assistant  Chief:  A.  M.  Fabbington. 

Chief  Clerk:  Charles  C.  Cabboll. 

Animal  Husbandry  Division:  Gkobge  M.  Rommel,  chief. 

Biochemic  Division:  M.  Dorset,  chief. 

Dairy  Division:  B.  H.  Rawl,  chief. 

Field  Inspection  Division:  R.  A.  Ramsay,  chief. 

Meat  Inspection  Division:  Rice  P.  Steddom,  chief. 

Pathological  Division:  John  R.  Mohleb,  chief. 

Quarantine  Division:  Richabd  W.  Hickman,  chief. 

Zoological  Division:  B.  H.  Ransom,  chief. 

Experiment  Station:  E.  C.  Schboedeb,  superintendent 

Editor:  James  M.  Pickens. 

DAIRY  DIVISION. 

B.  H.  Rawl,  chief. 

Helmeb  Rabild,  in  charge  of  Dairy  Farming  Investigations. 
S.  C.  Thompson,  in  charge  of  Dairy  Manufactunng  Investigations. 
L.  A.  RoGEBS,  in  charge  or  Research  Laboratories. 
Ebnest  Kelly,  in  charge  of  Market  Milk  Investigations. 
RoBEBT  McAdam,  in  charge  of  Renovated  Butter  Inspection. 
2 


ADDITIONAL  COPIES  of  this  publication 
2\.  may  be  procured  from  the  Supebintend- 
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Office,  Washington,  D.  C,  at  10  cents  per  copy 


LETTER  OF  TRANSMITTAL. 


U.  S.  Department  of  Agriculture, 

Bureau  of  Animal  Industry, 
Washington,  D.  C,  May  ^4,  191^. 

Sir:  I  have  the  honor  to  transmit  for  publication  in  the  bulletin 
series  of  the  bureau  the  accompanying  manuscript  entitled  "  The 
Bacteriology  of  Cheddar  Cheese,"  by  Messrs.  E.  G.  Hastings  and 
E.  B.  Hart,  of  the  Wisconsin  Agricultural  Experiment  Station,  and 
Miss  Alice  C.  Evans,  of  the  Dairy  Division  of  this  bureau. 

Cooperative  work  on  the  factors  concerned  in  the  ripening  of 
Cheddar  cheese  has  for  several  years  been  carried  on  at  Madison, 
Wis.,  between  the  Dairy  Division  and  the  Wisconsin  Experiment 
Station,  and  this  bulletin  is  one  of  a  series  in  which  the  results  of 
the  work  are  detailed.  Previous  experiments  have  already  been  de- 
scribed in  Bulletin  122  of  the  Bureau  of  Animal  Industry,  entitled 
"  Factors  Controlling  the  Moisture  Content  of  Cheese  Curds,"  and 
in  Bulletins  7  and  11  of  the  Wisconsin  Station. 

In  connection  with  the  present  study  credit  is  due  to  Mr.  L.  D. 
Bushnell,  now  with  the  Kansas  Agricultural  Experiment  Station, 
and  Mr.  Alfred  Larsen,  of  the  North  Dakota  State  Hygiene  Labora- 
tory, who  were  formerly  bacteriologists  in  the  Dairy  Division  and 
while  thus  engaged  prepared  a  portion  of  the  data  presented  in  this 
bulletin. 

KespectfuUy,  A.  D.  Melvin, 

Chief  of  Bureau. 

Hon.  James  Wilson, 

Secretary  of  Agriculture, 

8 


Digitized  by  the  Internet  Archive 

in  2007  with  funding  from 

IVIicrosoft  Corporation 


http://www.archive.org/details/bacteriologyofchOOhastiala 


CONTENTS. 


Page. 

Introduction 7 

The  bacterial  flora  of  milk 8 

Differences  in  composition  of  cheese 9 

The  enzym  content  of  cheese 10 

Biological  problems 10 

Bacteriological  methods 11 

The  role  of  Bacterium  lactis  acidi  in  Cheddar  cheese 13 

Effect  of  curdling  on  distribution  of  bacteria  in  milk 15 

Effect  of  acid  on  the  expulsion  of  the  whey 18 

Effect  of  acid  on  the  texture  of  the  curd 18 

The  role  of  acid  in  the  ripening  of  cheese 18 

The  protective  action  of  acid 20 

The  rate  of  bacterial  growth  in  cheese 21 

Methods  of  examination * 21 

Results  of  the  work 22 

The  enzymic  action  of  lactic  bacteria 25 

Other  groups  of  bacteria  in  Cheddar  cheese 31 

Determinations  by  milk-dilution  and  plate-culture  methods 31 

Determinations  by  microscopic  examination  of  the  cheese 37 

The  disappearance  of  bacterial  cells  in  cheese 39 

Detailed  study  of  lactic  bacilli 40 

Acidity  produced 41 

Forma  of  lactic  acid  produced 41 

Type  of  curd  produced 42 

Fermentation  of  sugars 43 

Form  of  colonies 43 

Thermal  death  point 44 

Morphology 44 

Conditions  for  growth  in  cheese 45 

Solvent  effect  on  milk  proteins 47 

Coccus  forma  in  Cheddar  cheese 48 

Chromogenic  cocci 50 

Liquefying  organisms 50 

The  sequence  in  development  of  bacterial  groups  in  Cheddar  cheese 51 

Summary 52 

5 


ILLUSTRATIONS 


PLATE. 


Page. 


Plate  I.  Fig.  1.— The  action  of  rennet  extract  on  casein  suspended  in  agar  in 
the  presence  and  in  the  absence  of  acid-forming  bacteria.    Fig. 
2. — ^The  effect  of  acid  on  cheese 20 

TEXT  FIGURES. 

Figure  1.  Increase  of  acidity  in  raw  milk  preserved  with  3  per  cent  toluol 29 

2.  Increase  of  acidity  in  heated  milk  preserved  with  3  per  cent  toluol.        29 

3.  Typical  microscopic  fields  from  cheese  No.  54  at  different  stages  in 

the  ripening  process 38 

4.  Typical  microscopic  fields  from  cheese  No.  91  at  different  stages  in 

the  ripening  process 39 

6 


THE  BACTERIOLOGY  OF  CHEDDAR  CHEESE. 


INTRODUCTION. 

The  role  of  microorganisms  in  the  preparation  and  ripening  of 
the  various  kinds  of  cheese  is  a  problem  that  has  attracted  the  at- 
tention of  bacteriologists  since  the  beginning  of  the  science  and  since 
the  establishment  of  the  importance  of  microorganisms  in  all  de- 
composition processes.  From  the  same  raw  materials — cow's  milk, 
salt,  and  rennet,  which  is  an  extract  of  the  fourth  stomach  of  the 
young  calf — a  great  number  of  varieties  of  cheese  are  prepared. 
These  cheeses  differ  not  only  in  texture,  but  more  especially  in  flavor 
and  aroma.  From  what  is  known  of  the  importance  of  microorgan- 
isms in  the  preparation  of  the  products  of  the  fermentation  indus- 
tries it  is  evident  to  one  who  gives  consideration  to  the  question  that 
undoubtedly  microorganisms  are  as  essential  in  the  making  of  cheese 
as  they  are  known  to  be  in  the  preparation  of  wine,  beer,  and  other 
products. 

In  the  making  of  wine  and  beer  the  desired  changes  are  produced 
by  a  single  form  of  life,  the  true  yeasts,  and  in  the  preparation  of 
any  desired  type  of  product  attention  need  only  be  directed  as  far 
as  the  causal  organism  is  concerned  in  order  to  insure  the  presence 
of  the  particular  variety  of  yeast  that  has  been  found  by  experience 
to  form  a  product  that  has  the  desired  properties  as  well  as  to 
insure  the  absence  of  harmful  forms.  In  the  preparation  of  still 
other  products  of  the  fermentation  industries  not  only  a  single  form 
of  life  is  essential  to  produce  the  desired  changes,  but  two  or  more 
are  needed,  one  of  which  may  work  on  a  certain  constituent  of  the 
raw  material,  the  other  on  another,  or  the  one  may  serve  to  make 
conditions  favorable  for  the  growth  of  the  second,  usually  by  the 
preparation  of  suitable  food. 

It  is  evident  that  as  the  number  of  kinds  of  microorganisms  that 
are  needed  to  produce  the  desired  changes  in  any  product  increases, 
the  complexity  of  the  problem  of  demonstrating  the  role  of  each 
of  them  is  also  greatly  increased,  and  it  may  become  most  difficult  to 
prove  even  whether  any  particular  organism  is  absolutely  essential 
in  the  preparation  of  the  product,  to  say  nothing  of  demonstrating 
its  exact  role.    It  is  not  to  be  supposed  that  a  single  form  of  life  is 

7 


8  BACTERIOLOGY  OF   CHEDDAR   CHEESE. 

responsible  for  the  complex  chemical  chancres  that  occur  in  the  ripen- 
ing of  any  particular  variety  of  cheese,  but  rather  that  a  considerable 
number  will  be  involved. 

The  fermentation  industries  include  those  in  which  microorganisms 
are  essential  for  the  production  of  the  desired  decomposition  changes. 
In  all  such  industries  the  quality  of  the  product  will  depend  on  the 
raw  material  and  the  organisms  that  are  used  in  the  decomposition  of 
that  material.  The  preparation  of  cheese  has  not  until  recent  years 
been  classed  as  a  fermentation  industry,  although  it  is  one  in  which 
microorganisms  play  a  dominant  role.  Here,  as  in  all  other  similar 
industries,  if  control  of  the  quality  of  the  product  is  to  be  obtained, 
knowledge  must  be  gained  concerning  the  essential  biological  agents. 
In  this  paper  are  presented  a  summary  of  the  present  knowledge  of 
the  bacteriology  of  Cheddar  cheese  and  the  results  of  a  detailed  study 
of  a  number  of  cheeses. 

THE  BACTERIAL  FLORA  OF  MILK. 

In  the  case  of  many  of  the  products  of  the  fermentation  industries 
the  essential  microorganisms  are  contained  in  the  raw  materials,  and 
the  development  of  the  methods  of  preparation  of  the  products  have 
been  wholly  empirical.  Especially  is  this  true  of  certain  kinds  of 
cheese,  the  methods  of  manufacture  of  which  are  in  some  cases  cen- 
turies old.  Even  in  the  case  of  cheese  to  which  at  some  stage  in 
manufacture  material  containing  certain  microorganisms  is  added  the 
methods  of  manufacture  antedate  the  science  of  bacteriology. 

The  sources  from  which  microorganisms  enter  the  milk  are  much 
the  same  in  all  parts  of  the  world.  Any  natural  source,  such  as  the 
interior  of  the  udder,  the  dust  from  the  animal,  or  even  the  utensils, 
will  always  furnish  certain  definite  types  of  microorganisms.  By 
this  is  not  meant  that  the  flora  of  milk  will  always  include  these 
definite  types  and  no  others,  but  that  certain  forms  will  always  be 
present.  That  this  is  true  is  shown  by  the  fact  that  a  sample  of 
ordinary  market  milk,  if  stored  at  temperatures  ranging  from  60° 
to  90°  F.,  will  undergo  a  definite  sequence  of  decomposition  changes 
with  almost  unerring  certainty.  In  this  sequence  three  main  groups 
of  microorganisms  are  prominent;  first,  the  acid-forming  bacteria 
that  change  the  milk  into  an  acid,  semisolid  mass,  favorable  for  the 
development  of  the  mold  that  is  characteristic  of  milk — Oidium  lactis. 
This  mold,  by  its  gradual  destruction  of  the  acid  and  the  establish- 
ment of  an  alkaline  reaction,  makes  it  possible  for  the  putrefactive 
organisms  to  develop.  When  the  environment  is  changed  this 
sequence  of  microbial  life  in  the  milk  is  also  changed.  For  example, 
milk  may  be  kept  at  such  low  temperatures  that  the  acid-forming 
bacteria  can  not  grow  and  the  putrefactive  bacteria  appear  first. 


DIFFERENCES  IN   COMPOSITION   OF   CHEESE.  9 

It  is  thus  proper  to  speak  of  a  characteristic  milk  flora.  By  some 
it  is  believed  that  this  flora  is  so  constant  that  fermentations  that 
depart  from  the  normal  are  to  be  looked  upon  not  as  due  to  the  in- 
troduction of  other  forms  of  microorganisms,  but  most  often  to 
changes  in  the  properties  of  the  organisms  or  to  changes  in  environ- 
ment that  favor  the  particular  organisms  which  produce  the  abnor- 
mal fermentation.  Slight  changes  in  the  composition  of  the  milk 
may  also  have  a  determining  influence. 

As  has  been  stated,  the  microorganisms  necessary  for  the  prepara- 
tion of  most  kinds  of  cheese  are  contained  in  the  milk.  Whether  one 
form  or  the  other  is  to  develop  in  the  cheese  depends  upon  the  en- 
vironment established  by  the  cheese  maker.  The  manufacture  of 
cheese  is  thus  a  problem  in  the  ecology  of  microorganisms. 

DIFFERENCES   IN    COMPOSITION    OF   CHEESE. 

The  differences  due  to  the  methods  of  manufacture  result  in  differ- 
ences in  composition  of  the  cheese  at  the  time  it  begins  to  undergo 
the  ripening  process,  which  is  to  determine  whether  the  cheese  is  to 
be  classed  as  one  kind  or  another.  The  differences  in  composition 
of  the  fresh  cheese  are  very  largely  in  the  amount  of  moisture  pres- 
ent. Since  the  water  that  is  left  in  the  cheese  is  whey,  carrying  in 
solution  sugar  and  other  constituents  of  milk,  the  effect  of  differ- 
ences in  moisture  is  not  only  in  influencing  the  texture,  but  also  the 
composition  of  the  cheese.  The  difference  in  moisture  content 
between  Cheddar  and  Camembert  cheese  is  approximately  25  per 
cent.  If  it  is  supposed  that  the  moisture  of  the  cheese  will  have 
about  the  same  sugar  content  as  milk,  this  means  that  the  sugar  con- 
tent of  the  Camembert  cheese  will  be  about  1  per  cent  greater  than 
Cheddar;  and  since  the  sugar  is  all  fermented,  differences  in  sugar 
content  ultimately  result  in  differences  in  acidity. 

One  of  the  basal  differences  between  Cheddar  and  Swiss  cheese  is 
that  during  the  making  of  the  first — that  is,  the  operations  before 
pressing — a  large  amount  of  acid  is  formed  in  the  curd,  while  in  the 
case  of  Swiss  no  such  acid  production  occurs.  Van  Slyke  and  Hart,^ 
Boekhout  and  Ott  de  Vries,^  and  Van  Dam^  have  shown  that  acid  is 
held  chemically  by  the  proteins  of  cheese,  while  sugar  is  not.  Thus 
if  in  the  making  of  one  cheese  acid  is  formed,  and  in  another  not 
formed,  the  result  will  be  a  somewhat  different  composition,  even 
though  the  moisture  content  of  the  two  cheeses  is  initially  the  same. 

1  Van  Slyke,  L.  L.,  and  Hart,  E.  B.  A  study  of  some  of  the  salts  formed  by  casein  and 
paracasein  with  acids :  Their  relations  to  American  Cheddar  cheese.  New  York  Agricul- 
tural Experiment  Station  Bulletin  214.     Geneva,  .Tuly,  1902.     See  p.  60. 

»  Boekhout,  F.  W.  J.,  and  Ott  de  Vries,  J.  .1.  ijber  den  Kiisefehler  "  Kurz  "  (Kort). 
Centralblatt  fUr  Bakteriologie,  Parasltenkunde  und  Infektlonskrankheiten,  Alrtellung  2, 
vol.  24,  No.  5/7,  pp.  122-129.     Jena,  .\ug.  2,  1909. 

«  Van  Dam,  W.  ijber  die  Konsistenz  dcr  KUsemasse  bel  EdamkSsen.  Centralblatt  fUr 
Bakteriologle,  Parasltenkunde  und  Infektlonskrankheiten,  Abteilung  2,  vol.  32,  No.  1/2, 
pp.  7-40.     Jena,  Dec.  5.  1911. 

50978°— Bull.  150—12 2 


10  BACTERIOLOGY   OF   CHEDDAR   CHEESE. 

Another  difference  in  composition  of  cheese  is  caused  by  variations 
in  the  amount  of  salt  and  in  the  time  at  which  it  is  added.  This  dif- 
ference is  an  important  one  in  the  case  of  Cheddar  and  Swiss  cheese. 
These  diSerences  in  the  initial  composition  are  sufficient  to  exert  a 
great  influence  on  the  types  of  life  that  are  to  grow  in  and  on  the 
cheese. 

THE  ENZYM  CONTENT  OF  CHEESE. 

The  role  of  the  inherent  enzyms  of  milk  and  those  of  the  reimet 
extract  in  the  ripening  of  cheese  have  been  emphasized  by  the  work 
of  Babcock  and  Russell  and  of  Von  Freudenreich  and  Jensen.  There 
remains  no  doubt  concerning  the  importance  of  certain  enzyms,  es- 
pecially those  of  rennet  extract,  in  the  ripening  of  cheese.  The 
enzymic  content  of  all  cheese  must  be  much  the  same  qualitatively, 
since  they  are  made  from  the  same  raw  materials.  Again,  it  is  dif- 
ficult to  see  how  one  enzym  can  be  active  in  one  cheese  and  not  in 
other  kinds,  since  the  conditions  are  quite  similar  as  regards  reaction 
and  other  factors.  Accepting  the  theory  of  the  specificity  of  enzyms, 
it  must  be  admitted  that  the  products  of  the  activity  of  any  enzym 
will  be  the  same  in  kind  no  matter  what  the  conditions  under  which 
it  may  be  working.  Hence  it  does  not  seem  that  the  enzyms  of  milk 
or  rennet  can  be  factors  determining  the  kind  of  cheese  to  be  made 
by  any  method  from  the  ordinary  raw  materials,  but  that  the  deter- 
mining factors  must  be  biological. 

BIOLOGICAL   PROBLEMS. 

The  methods  of  manufacture  of  cheese  result  in  an  environment 
that  causes  certain  of  the  organisms  constantly  present  in  milk  to 
develop  in  a  definite  sequence.  The  empirical  methods  of  the  cheese- 
maker  result  in  a  cheese  that  approximates,  if  it  is  not  identical  with, 
the  normal  of  its  kind. 

Each  of  the  great  commercial  varieties  of  cheese  thus  presents  a 
distinct  problem  for  the  bacteriologist,  who  should  be  able  to  demon- 
strate the  constant  presence  of  the  essential  groups  of  bacteria.  It 
can  not  be  expected  here  any  more  than  in  most  decomposition  proc- 
esses that  a  single  specific  organism  will  be  concerned,  as  in  the 
causation  of  disease,  but  rather  that  groups  of  organisms  will  be 
present,  the  basis  of  grouping  being  largely  biochemical.  Neither 
can  it  be  expected  that  one  group  will  grow  for  a  period,  then  dis- 
appear and  be  followed  by  a  second,  but  rather  that  the  sequence  of 
development  wijl  be  confused. 

The  work  of  Thorn  on  Camembert  cheese  furnishes  an  excellent 
illustration.  It  has  been  shown  that  a  definite  balance  between  the 
various  forms  of  life  concerned  is  essential  for  the  normal  ripening 


BACTERIOLOGICAL  METHODS.  11 

of  this  cheese.  If  through  methods  of  manufacture  or  through 
conditions  of  ripening  this  balance  is  destroyed,  the  cheese  will  not 
be  typical. 

The  biological  factors  concerned  in  the  ripening  of  Camembert 
cheese  are  the  acid-forming  bacteria,  the  Camembert  mold  {Penicil- 
lium  camemherti) ,  or  its  white  form  {PeniciUium  camemherti  var. 
rogeri) ;  Oidium  lactis;.  and  bacteria  which  together  with  Oidium 
lactis  form  a  reddish  slime  on  the  surface  of  the  cheese.  If  condi- 
tions favor  the  growth  of  Oidium  lactis  at  the  expense  of  the  penicil- 
lium,  the  cheese  will  not  be  normal;  if  the  mold  is  too  luxuriant, 
texture  is  obtained  but  flavor  is  not.  It  is  thus  evident  in  the  case  of 
Camembert  cheese  too  wide  variations  in  the  balance  between  the 
different  forms  of  life  can  not  occur  if  a  cheese  normal  in  texture 
and  flavor  is  to  be  obtained.  A  large  amount  of  work  has  been  done 
on  Camembert  cheese  and  the  necessity  for  the  presence  of  the  dif- 
ferent forms  established,  but  their  exact  role  has  not  been  shown  and 
never  can  be  until  the  chemist  devises  means  of  following  in  detail 
the  complex  chemical  changes. 

It  is  not  only  necessary  that  the  constant  presence  of  any  organism 
in  cheese  be  demonstrated  in  order  to  prove  its  importance  in  the 
ripening,  but  it  must  also  be  present  in  sufficient  numbers  so  that 
it  is  certain  that  growth  has  taken  place  in  the  cheese.  The  simple 
presence  of  any  form  is  no  evidence  of  its  activity.  Such  an  error 
was  made  by  Duclaux,  one  of  the  first  bacteriologists  to  occupy  him- 
self with  cheese  problems.  Since  there  are  such  a  multitude  of  forms 
of  bacteria  in  milk,  and  hence  in  cheese,  any  particular  method  of 
examination  of  the  cheese  is  likely  to  bring  some  one  group  of 
organisms  to  the  front.  The  methods  used  by  Duclaux  in  his  examir 
nation  of  Cantal  cheese  were  fitted  to  favor  the  development  of  the 
spore-bearing,  liquefying  bacteria.  Their  constant  presence,  together 
with  the  fact  that  they  produced  in  pure  culture  in  milk  compounds 
which  he  had  already  demonstrated  in  the  ripe  cheese,  led  him  to 
consider  them  an  important  factor  in  the  ripening  of  this  cheese.  It 
has  been  demonstrated  since  that  the  conditions  in  hard  cheese  do 
not  permit  this  class  of  bacteria  to  develop. 

BACTERIOLOGICAL  METHODS. 

The  modern  bacteriological  methods  are  not  such  as  will  demon- 
strate the  presence  of  all  kinds  of  bacteria  in  milk,  cheese,  or  any 
other  substance  in  which  a  considerable  number  of  kinds  of  bacteria 
are  present,  even  though  all  may  find  conditions  that  will  permit  of 
growth  on  the  medium  employed.  It  is  usually  easy  to  demonstrate 
the  presence  of  the  form  that  occurs  in  greatest  numbers,  since  in 
the  more  lightly  inoculated  culture  plates  this  form  will  be  present 


12  BACTERIOLOGY   OF   CHEDDAR   CHEESE. 

in  pure  culture  or  else  so  freed  from  competition  with  the  other 
forms  that  its  growth  will  not  be  prevented.  For  those  forms  present 
in  less  numbers  this  condition  does  not  obtain,  and  usually  special 
methods  must  be  employed  to  demonstrate  their  presence,  such  as 
the  use  of  differentiating  media  or  enrichment  cultures.  Anyone 
who  has  much  experience  in  plating  milk  on  lactose  media  has  noted 
that  in  certain  cases  the  plates  thickly  seeded  show  colonies  of  but 
a  single  organism,  usually  the  ordinary  lactic  organism  Bacterium 
lactis  acidi,  while  on  the  more  thinly  seeded  plates  from  the  same 
sample  other  forms  may  appear.  In  the  thickly  seeded  plates  the 
lactic  organisms,  on  account  of  favorable  conditions,  have  grown 
most  rapidly  and  by  the  acid  produced  have  prevented  the  develop- 
ment of  other  forms,  while  in  the  more  thinly  seeded  plates  the 
colonies  may  be  separated  by  such  a  distance  that  the  products  of  one 
colony  do  not  reach  the  others,  each  colony  then  grows  as  though 
it  were  the  only  colony  on  the  plate.  Hence  forms  appear  on  thinly 
seeded  plates  that  do  not  on  those  crowded  with  colonies.  If  the 
organism  that  finds  most  favorable  conditions  greatly  exceeds  in 
numbers  some  other  form  the  latter  may  never  appear  on  the  cul- 
ture or  be  so  inconstant  as  not  to  attract  the  attention  of  the  bac- 
teriologist. A  ratio  of  1,000  to  1  or  even  500  to  1  may  prevent  the 
detection,  by  use  of  the  ordinary  plate-culture  methods,  of  the 
organisms  present  in  smaller  numbers.  This  statement  may  serve 
to  explain  the  results  obtained  by  previous  investigators  of  Cheddar 
cheese. 

Before  1896  no  consecutive  bacteriological  examinations  had  been 
made  of  a  ripening  Cheddar  cheese.  The  first  detailed  work  on  this 
variety  of  cheese  with  which  the  writers  are  acquainted  is  that  of 
Russell.^  This  work  was  largely  concerned  with  a  quantitative  exam- 
ination of  the  cheese  during  the  ripening  process  by  means  of  gelatin 
plates.  It  established  the  fact  that  acid-forming  organisms  make  up 
99  per  cent  and  over  of  the  bacteria  thus  determined.  It  may  be 
inferred  that  the  acid-forming  bacteria  belonged  to  the  Bacterium 
lactis  acidi  group.  The  work  of  Lloyd  on  English  Cheddar  cheese 
led  to  similar  results.  The  most  extensive  work  on  the  bacterial 
flora  of  Cheddar  cheese  is  that  of  Harding  and  Prucha,^  who  iso- 
lated the  different  types  of  organisms  appearing  on  cultures  made 
from  9  normal  Cheddar  cheeses  during  the  entire  period  of  ripening. 
The  more  than  300  cultures  thus  isolated  were  studied  in  detail  and 
reduced  to  33  groups.  Of  these  33  groups  4  belonged  to  the  Bacte- 
rium, lactis  acidi  group,  the  only  one  which  was  always  found,  and 
it  practically  always  included  over  99  per  cent  of  the  total  germ 

*  Russell,  II.  L.  The  rise  and  fall  of  bacteria  In  Cheddar  cheese.  Wisconsin  Agricul- 
tural Experiment  Station,  Thirteenth  Annual  Report,  pp.  95-111.     Madison,  1896. 

•  Harding,  II.  A.,  and  Prucha,  M.  J.  The  bacterial  flora  of  Cheddar  cheese.  New 
York  Agricultural  Experiment  Station,  Technical  Bulletin  8.     Geneva,  Dec,  1908. 


THE  BOLE  OP  BACTERIUM   LACTIS  ACIDI.  13 

content.  The  10  other  groups  which  these  investigators  classed  as 
important  on  account  of  frequency  of  occurrence  were  not  found  in 
all  of  the  cheeses,  and  it  may  be  inferred  that  they  do  not  represent 
essential  factors  in  the  ripening  of  Cheddar  cheese.  In  speaking  of 
one  cheese  the  authors  say : 

The  results  from  this  cheese  accord  with  the  idea  that  aside  from  tlie  lactic 
group  there  is  no  single  group  or  at  least  no  single  species  of  bacteria  abso- 
lutely essential  to  the  ripening  process. 

The  work  of  previous  investigators  has  shown  that  the  liquefying, 
the  gas-formingj  and  the  inert  bacteria  are  not  essential  factors  in 
the  ripening  of  Cheddar  cheese,  since  all  or  any  one  of  these  groups 
may  be  absent  and  yet  the  cheese  may  ripen  in  a  normal  manner. 
It  is  true  that  all  of  these  groups  are  usually  represented  in  Cheddar 
cheese,  since  they  are  present  in  milk,  but  the  numbers  are  small, 
and  in  the  case  of  the  liquefying  organisms  there  is  no  evidence  that 
growth  ever  occurs  during  the  ripening  process. 

Yeasts  can  not  be  classed  as  an  important  factor  since  they  may  be 
absent  from  a  normal  cheese. 

The  work  of  previous  investigators  may  be  summarized  in  the 
statement  that  the  group  of  bacteria  represented  by  Bacterium  lactis 
acidi  is  the  only  one  that  up  to  the  present  has  been  shown  to  be  of 
constant  occurrence  in  Cheddar  cheese.  This  fact- together  with  the 
enormous  numbers,  amounting  to  millions  and  at  times  over  a  billion 
per  gram  of  the  fresh  cheese,  leaves  no  doubt  of  the  importance  of 
this  group  of  organisms  in  Cheddar  cheese,  and  undoubtedly  in  all 
cheese  that  undergoes  a  ripening  process. 

THE   ROLE   OF  BACTERIUM    LACTIS  ACIDI  IN  CHEDDAR  CHEESE. 

The  empirical  methods  of  the  Cheddar  cheesemaker  demand  a  milk 
so  far  advanced  in  the  acid  fermentation  that  during  the  few  hours 
between  time  of  curdling  and  placing  the  curd  in  the  press  acid  de- 
velopment is  rapid.  The  increasing  acidity,  as  has  long  been  known, 
favors  the  curdling  of  the  milk  by  the  rennet.  Indeed  the  tests  most 
frequently  used  in  cheesemaking  to  determine  the  "  ripeness "  or 
acidity  of  the  milk  are  those  in  which  the  time  of  curdling  is  noted, 
when  a  definite  amount  of  rennet  is  added  to  milk  at  a  definite  tem- 
perature (tests  of  Monrad  and  Marschall).  The  Cheddar  maker 
desires  milk  that  has  just  passed  through  the  "  period  of  incubation." 
By  this  expression  is  meant  the  time  during  which  no  apparent  in- 
crease in  acidity  results  from  the  growth  of  acid-forming  bacteria. 
This  has  been  supposed  to  be  due  to  the  fact  that  no  acid  was  formed 
by  the  rapidly  growing  organisms.  More  recently  it  has  been  pointed 
out  by  Rahn  ^  that  this  is  not  the  true  explanation  but  that  until  the 

iRahn,  Otto.  The  fermenting  capacity  of  the  average  individual  cell  {Bacterium 
lactis  acidi).    Science,  now  series,  vol.  33,  No.  849,  pp.  539-540.    New  York,  Apr.  7,  1911. 


14 


BACTEEIOLOGY  OF   CHEDDAE  CHEESE, 


bacteria  have  increased  to  a  great  extent  the  amount  of  acid  formed 
is  so  small  as  to  escape  detection.  Whatever  the  explanation,  it  is 
true  that  the  acid-forming  bacteria  may  increase  until  there  are 
millions  per  cubic  centimeter  and  yet  the  acidity  show  no  change 
whatever,  as  has  been  shown  by  Koning  ^  and  by  Burri  and 
Kiirsteiner.^ 

At  last  the  acidity  begins  to  develop  with  increasing  rapidity  until 
it  reaches  a  point  where  it  begins  to  exert  an  inhibitive  effect  upon  the 
growth  of  the  organism.  In  Table  1  are  given  some  data  illustrating 
the  rate  of  acid  development  in  milk  kept  at  95°  F.  It  will  be  noted 
in  both  samples  that  the  initial  rate  of  increase  in  acid  is  small,  that 
it  reaches  a  maximum  that  may  be  over  0.15  per  cent  per  hour  and 
then  declines.  Both  samples  of  milk  were  slightly  too  acid  for 
cheesemaking. 

Table  1. — Rate  of  development  of  acid  in  milk  kept  at  95°  F. 


Hours. 


Sample  1: 

Acidity , 

Increase  per  hour. 
Sample  2: 

Acidity 

Increase  per  hom-. 


Per  cent. 
0.24 


.20 


Per  cent. 

0.30 

.06 

.24 
.04 


Per  cent. 

0.40 

.10 

.34 
.10 


Per  cent. 

0.47 

.07 

.5 
.16 


Per  cent. 

0.71 

.12 

.74 
.12 


Per  cent. 
1.00 
.015 


.012 


This  rapid  increase  in  acidity  is  the  result  of  the  growth  of  the 
acid-forming  bacteria  in  the  favorable  environment.  In  Table  2  are 
given  the  results  of  the  bacteriological  examination  of  two  samples  of 
milk  kept  at  95°  F.  for  a  number  of  hours.  It  will  be  noted  that  the 
increase  in  numbers  of  bacteria,  like  that  of  the  acidity,  goes  on  with 
increasing  rapidity  until  a  maximum  is  reached,  after  which  the 
growth  is  less  and  less  rapid. 

Table  2. — Increase  o/  bacteria  per  cubic  centimeter  in  milk  kept  at  95°  F. 

[Number  of  bacteria  expressed  in  millions.] 


Sample  1. 
Sample  2. 


Hours. 


83 


231 
157 


927 


1,064 
1,545 


10 


1,167 
1,982 


'  Konlnj?,  C.  J.  Der  Sfturojirad  dor  Milch,  rpharmaceutisch  Weekblad,  1904]  Mllch- 
wirtshaftliches  Zentralblatt,  vol.  1,  No.  7,  pp.  289-305,  July;  No.  8,  pp.  337-356, 
.August.     Leipzig,  1905.     See  p.  294. 

-  Rurri.  R.,  and  Kllrsteiner,  .T.  UntcrRUcliunfron  fiber  dip  Relfung  der  Kilserelmllch. 
LandwirtBCbaftlicbes  Jabrbuch  der  Scbweiz,  vol.  24,  pp.  437-466.     Bern,  1910. 


EFFECT  OF   CURDLING. 


15 


It  is  essential  that  the  acid  development  should  be  rapid  during  the 
time  between  the  curdling  of  the  milk  and  pressing  the  curd.  The 
modern  Cheddar  maker  insures  this  by  the  addition  of  large  numbers 
of  acid-forming  bacteria  in  the  form  of  a  starter. 

EFFECT   OF  CURDLING   ON   DISTRIBUTION   OF   BACTERIA   IN    MILK. 

The  solid  bodies  present  in  the  milk  will  be  held  by  the  curd  in  the 
same  manner  as  the  formation  of  aluminum  or  ferric  hydrate  in  water 
enmeshes  the  solid  bodies  present,  or  the  coagulating  of  albumen  in 
a  solution  removes  turbidity  therefrom,  as  in  the  clearing  of  bac- 
teriological media  and  of  wine. 

In  order  to  illustrate  the  effect  of  curdling  on  the  distribution  of 
bacteria  several  samples  of  milk  were  subjected  to  a  quantitative  ex- 
amination. Rennet  solution  was  then  added  and  the  curd  cut.  As 
soon  as  possible  a  sample  of  the  whey  was  likewise  examined.  In 
Table  3  are  given  the  data  obtained  from  a  number  of  such  examina- 
tions. It  will  be  noted  that  in  all  cases  a  unit  volume  of  the  whey 
contained  less  bacteria  than  the  milk  before  curdling.  From  the 
average  figures  of  the  determinations  approximately  77  per  cent  of 
the  bacteria  were  retained  in  the  curd  in  the  trials  made  in  the 
laboratory. 

Table  3. — Numhcr  of  bacteria  per  cubic  centimeter  in  milk  and  in  v^hey  im- 
mediately after  curdling — Laboratory  experiments. 

[Number  of  bacteria  expressed  In  thousands.) 


Trial  No. 

1 

2 

3 

4 

5 

6 

MUk 

128,000 
34,000 

11,000 
2,900 

2,050 
730 

155,000 

750) 

2;?,  000 
773 

59,000 

Whey 

776 

A  number  of  similar  examinations  were  made  under  practical  con- 
ditions in  the  cheese  room,  the  sample  of  milk  being  taken  from  the 
cheese  vat  and  the  whey  after  cutting  the  curd  in  the  usual  manner. 
The  data  are  given  in  Table  4.  It  will  be  noted  that  the  results  are 
similar  to  those  obtained  in  the  laboratory.  Approximately  73  per 
cent  of  the  bacteria  in  the  milk  were  retained  in  the  curd. 

Table  4. — Number  of  bacteria  per  cubic  centimeter  in  milk  and  in  tvlicy  itn- 
mediately  after  curdling — Samples  from  the  cheese  vat. 

[Number  of  bacteria  expressed  in  thousands.] 


Trial  No. 

1 

2 

3 

4 

5 

Milk 

6,600 
3,000 

6,400 
1,290 

5.430 
2,400 

4,000 
210 

6,500 

Whey 

460 

16 


BACTEEIOLOGY   OF   CHEDDAR   CHEESE. 


The  result  of  curdling  and  the  shrinking  of  the  curd  is  that  the 
acid-forming  bacteria  of  the  milk  are  concentrated  in  the  curd,  which 
soon  after  cutting  occupies  but  a  fraction  of  the  volume  of  the  origi- 
nal milk.  This  concentration,  together  with  the  favorable  environ- 
ment, results  in  a  rapid  formation  of  acid  in  the  curd. 

This  condition  should  result  in  a  more  rapid  increase  of  acid  in  a 
whey  in  which  the  curd  is  allowed  to  remain  than  in  a  portion  of 
the  same  whey  removed  from  the  curd.  As  the  curd  shrinks  the 
expelled  whey  should  bring  with  it  a  portion  of  the  acid  that  has 
been  formed  in  the  curd;  this,  together  with  osmotic  action,  should 
increase  the  acidity  of  the  whey  in  contact  with  the  curd. 

In  order  to  demonstrate  this  a  sample  of  milk  was  curdled,  the 
curd  cut,  and  as  soon  as  possible  a  portion  of  the  whey  removed  to 
a  separate  vessel.  Acidity  determinations  were  made  at  intervals. 
The  results  are  given  in  Table  5.  It  will  be  noted  that  in  every  case 
the  acidity  of  the  whey  in  contact  with  the  curd  increased  much 
more  rapidly  than  that  of  the  whey  not  in  contact  with  the  curd. 

Table  5. — Development  of  acid  in  tchey  alone  and  in  whey  in  contact  xcith  curd. 


Hours. 

0 

2 

4 

6 

8 

10 

12 

Sample  1 : 

Whey 

Percent. 

0.09 

.09 

.14 
.14 

.10 
.10 

.09 
.09 

Per  cent. 

0.09 

.10 

.20 
.27 

.12 
.12 

.10 
.10 

Per  cent. 

0.10 

.16 

.30 

.48 

.12 
.16 

.11 
.13 

Per  cent. 

0.13 

.17 

.35 
.53 

.22 
.32 

.13 
.18 

Per  cent. 

0.16 

.32 

.49 
.68 

.33 
.49 

.14 
.20 

Per  cent. 

0.24 

.47 

Per  cent. 
0.36 

W  hey  and  curd 

.54 

Sample  2: 

Whey             

"Whey  and  curd 

Sample  3: 

Whey 

W  hey  and  curd.. 

Sample  4: 

Whey 

.33 

.42 

Whey  and  curd 

It  was  thought  that  if  a  sample  of  milk  was  curdled  by  rennet  and 
the  curd  cut  and  allowed  to  settle  to  the  bottom  of  a  deep  container, 
the  whey  at  dijfferent  levels  should  show  varying  degrees  of  acidity 
if  the  container  were  so  protected  that  convection  currents  did  not 
tend  to  mix  the  liquid.  In  other  words,  the  acid  whey  expelled  would 
tend  to  accumulate  in  the  lower  portion  of  the  container. 

In  order  to  test  this,  deep  beakers  were  filled  with  milk,  the  milk 
curdled  with  rennet,  and  the  curd  cut  and  allowed  to  settle.  The 
beakers  were  kept  in  a  thermostat  at  95°  F. ;  thus  no  convection  cur- 
rents were  present,  since  care  was  taken  to  heat  the  milk  to  the  same 
temperature  before  curdling.  The  samples  of  whey  were  removed 
in  such  a  manner  as  not  to  mix  the  different  layers.  In  Table  6  the 
results  of  a  number  of  trials  are  given.  It  will  be  seen  that  the 
acidity  of  the  bottom  layers  increases  much  faster  than  that  of  the 
upper  layers,  the  difference  being  in  some  cases  0.4  per  cent. 


ACIDITY  OF   MILK. 


17 


Table  6. — Acidity  of  whey  at  the  top  and  bottom  of  a  vessel  containing  whey 

and  curd. 


Hours. 

0 

2 

4 

6 

8 

10 

12 

Sample  1: 

Top 

Percent. 

0.09 

.09 

.14 
.14 

.08 
.08 

.09 
.09 

Per  cent. 

0.09 

.10 

.21 

.34 

.08 
.08 

.10 
.10 

Per  cent. 

0.11 

.13 

.32 
.64 

.09 
.09 

.13 

.14 

Per  cent. 

0.12 

.22 

.36 

•n 

.09 
.11 

.14 

.22 

Percent. 

0.16 

.49 

.56 
.80 

.12 
.15 

.17 
.24 

Percent. 

0.27 

.67 

Percent. 
0.40 

Bottom 

.68 

Sample  2: 

Top 

Bottom 

Sample  3: 

Top 

.18 
.34 

.25 
.59 

.24 

Bottom.. 

.45 

Sample  4: 

Top 

Bottom 

It  is  thus  shown  in  a  number  of  ways  that  the  location  of  acid 
development  is  in  the  curd  rather  than  in  the  whey. 

It  has  been  shown  that  paracasein  wiU  absorb  and  probably  com- 
bine with  acids.  If  this  is  so,  a  portion  of  the  acid  formed  in  the 
curd  should  be  retained  in  loose  chemical  combination  and  the  acidity 
of  a  sample  of  milk  should  increase  more  rapidly  than  that  of  the 
whey  from  the  same  milk,  even  though  the  whey  is  in  contact  with 
the  curd.  The  data  given  in  Table  7  show  this  to  be  true.  The 
figures  represent  the  increase  in  acid  at  the  end  of  each  period  over 
the  initial  acidity  of  the  milk  or  whey  and  curd.  This  leaves  no 
doubt  that  a  portion  of  the  acid  is  retained  by  the  curd,  and  is  proof 
of  an  earlier  statement,  that  a  cheese  in  which  acid  is  developed  dur- 
ing the  process  of  making  will  have  a  different  acidity  from  one  with 
an  equal  content  of  moisture,  but  in  which  no  acid  is  formed  during 
the  making. 

Table  7. — Increase  in  acidity  of  milk  and  of  whey  in  contact  with  curd. 


Hours. 

2    • 

4 

6 

8 

10 

12 

Sample  1: 

Milk 

Per  cent. 

0.19 

.13 

.00 
.00 

.04 
.05 

.03 
.03 

Per  cent. 

0.34 

.32 

.01 
.01 

.21 
.11 

.15 
.04 

Per  cent. 

0.64 

.38 

.09 
.02 

.44 

.16 

.43 
.16 

Per  cent. 

0.73 

.52 

.32 
.05 

.69 
.28 

.63 
.25 

Per  cent. 

Per  cent. 

Sample  2: 

Milk 

0.55 
.18 

0.74 

.26 

Sample  3: 

MUk    .             

Sample  4: 

Milk  

50978"— Bull.  150—12- 


18  BACTERIOLOGY  OP   CHEDDAE  CHEESE. 

EFFECT  OF  ACID  ON  EXPULSION  OF  THE  WHEY. 

It  has  been  shown  by  Sammis,  Suzuki,  and  Laabs  ^  that  the  expul- 
sion of  whey  from  the  curd  is  directly  proportional  to  the  amount 
of  acidity  present;  at  least,  in  the  case  of  percentages  of  acidity  that 
are  met  with  in  normal  cheese  curds.  The  growth  of  lactic-acid 
bacteria  in  the  curd  and  the  consequent  development  of  acidity  are 
necessary  to  secure  a  curd  with  the  proper  moisture  content. 

EFFECT  OF  ACID  ON  THE  TEXTURE  OF  THE  CURD. 

The  curd  from  milk  in  which  no  growth  of  acid-forming  bacteria 
has  taken  place  shows  but  a  slight  tendency  to  mat  or  for  the  pieces 
of  curd  to  fuse.  If,  however,  acid  is  formed  in  the  pieces  of  curd, 
they  undergo  such  a  change  in  texture  that  as  soon  as  they  are  allowed 
to  settle  to  the  bottom  of  the  vat  they  soon  unite  to  form  a  single 
mass  of  curd.  In  the  making  of  cheese  by  the  Cheddar  process  it  is 
essential  that  this  matting  take  place. 

The  change  in  texture  which  the  curd  undergoes  is  due  to  the  action 
of  the  acid  on  the  paracasein,  forming  a  substance  that,  when  warmed, 
can  be  drawn  into  threads.  The  basis  of  the  "  hot  iron  "  test  as  used 
to  determine  the  time  to  draw  the  whey  is  the  formation  of  this 
compound  by  the  acid. 

At  the  time  the  curd  is  placed  in  the  press  there  are  numerous 
spaces  between  the  curd  particles.  It  is  essential  that  the  curd  be 
so  plastic  that,  under  the  influence  of  the  pressure,  the  particles 
undergo  perfect  fusion,  so  that  the  entire  cheese  is  one  mass,  per- 
fectly free  from  irregular-shaped  mechanical  holes.  Such  a  fusion 
does  not  occur  in  the  absence  of  acid  formation  in  the  curd. 

THE  r6LE  of  acid  IN  THE  RIPENING  OF  CHEESE. 

In  the  ripening  process  proper  the  acid  resulting  from  the  fer- 
mentation of  the  sugar  by  the  organisms  of  the  Bacterium,  lactia 
acidi  group  has  an  important  role.  As  has  been  shown  by  numerous 
investigators,  the  sugar  in  the  cheese  is  all  fermented  within  a  few 
days.  It  was  shown  by  Babcock  and  Eussell,^  also  by  Jensen,®  and 
by  Van  Slyke,  Harding,  and  Hart  *  that  rennet  extract  not  only  has 
a  curdling  effect  but  has  a  digestive  action  in  the  presence  of  an 

1  SammlB,  J.  L. ;  Suzuki,  S.  K.,  and  Laabs,  F.  W.  Factors  controlling  the  moisture 
content  of  cheese  curds.  U.  S.  Department  of  Agriculture,  Bureau  of  Animal  Industry, 
Bulletin  122.     Washington,  1910. 

*  Babcock,  S.  M. ;  Russell,  II.  L. ;  and  Vivian,  A.  Influence  of  rennet  on  cheese  ripen- 
ing. Wisconsin  Agricultural  Experiment  Station,  Seventeenth  Annual  Report,  pp.  102- 
122.     Madison,  1900. 

« Jensen,  Orla.  Studlen  (iber  die  Enzyme  Im  Kttse.  Landwirtschaftliches  Jahrbuch 
der  Schwclz,  vol.  14,  pp.  197-233.     Berne,  1900. 

*Van  Slyke,  L.  L. ;  Harding,  II.  A.;  and  Hart,  E.  B.  Rennet-enzyme  as  a  factor  in 
cheese  ripening.  New  York  Agricultural  Experiment  Station,  Bulletin  233.  Geneva, 
June,  1003. 


THE  ROLE   OF  ACID  IN  RIPENING  OF   CHEESE. 


19 


activating  acid  such  as  is  present  in  the  cheese.  The  rapid  proteoly- 
sis occurring  during  the  first  part  of  the  ripening  process  is  largely- 
due  to  the  action  of  the  pepsin. 

Variations  in  the  amount  of  rennet  extract  added  to  the  milk  result 
in  differences  in  the  rate  of  ripening,  as  has  been  shown  by  numerous 
investigators.  This  can  be  explained  only  through  the  variations  in 
the  pepsin  content  of  the  cheese.  The  action  of  rennet  extract  in 
the  presence  and  absence  of  lactic  organisms  is  demonstrated  in 
Plate  I,  figure  1.  A  4  per  cent  solution  of  agar  was  added  to  sterile 
milk  in  the  proportion  of  1  to  1.  A  portion  of  the  milk  agar 
was  heavily  inoculated  with  a  lactic  organism  and  the  plates  in- 
cubated for  24  hours;  the  other  portion  was  not  inoculated.  At  the 
end  of  the  period  of  incubation  strips  of  filter  paper  were  moistened 
with  rennet  extract  and  placed  on  the  plates,  which  were  then  in- 
cubated at  37°  C.  for  one  hour.  The  photograph  was  taken  at 
the  end  of  the  period.  It  will  be  noted  that  in  the  presence  of  the 
lactic  organisms  the  casein  has  been  rendered  soluble  by  the  rennet 
extract,  while  in  the  absence  of  the  organisms  no  such  marked 
digestion  has  occurred.  The  digestion  of  the  casein  has  resulted  in 
the  destruction  of  the  opacity  of  the  milk  agar  so  that  a  number 
placed  beneath  the  dish  can  be  read,  while  in  the  case  of  the  milk 
agar  from  which  the  lactic  organisms  were  absent  no  trace  of  a 
similar  number  can  be  distinguished.  In  the  milk  agar  conditions 
are  very  similar  to  those  obtaining  in  cheese,  the  activating  acid  being 
of  like  origin. 

It  would  thus  seem  that  if  a  cheese  was  made  from  milk  that  con- 
tained but  few  acid-forming  organisms  the  rate  of  ripening  would  be 
delayed.  In  order  to  test  this  hypothesis,  a  cheese  was  made  from 
very  clean  milk,  containing  scarcely  any  acid-producing  organisms, 
and  no  starter  was  added.  No  acid  whatever  was  formed  during  the 
making  process.  The  milk  after  standing  24  hours  at  20°  C.  had 
an  acidity  of  but  0.19  per  cent.  In  order  to  insure  proper  curdling, 
the  acidity  of  the  milk  was  raised  to  0.25  per  cent  by  the  addition 
of  hydrochloric  acid.  In  order  to  follow  the  rate  of  acid  formation, 
determinations  of  the  sugar  present  in  the  cheese  were  made  at 
interv^als.    The  results  are  given  in  Table  8. 

Table   8. — Sugar   content   of   cheese   made   from  milk   containing   few   lactic 

bacteria. 


Time. 

Sugar  con- 
tent. 

Acidity. 

Time. 

Sugar  con- 
tent. 

Acidity. 

Days. 

1 
2 
4 

PercerU. 
1.51 
1.49 
1.48 

Per  cent. 
.25 
.27 
.27 

Day. 

22 
46 

Per  cent. 

1.45 

.94 

.00 

PercerU. 

.27 

.45 

1.13 

20  BACTERIOLOGY  OF   CHEDDAR  CHEESE. 

The  sugar  disappears  from  a  normal  cheese  in  3  to  5  days,  while 
on  the  twenty-second  day  over  one-half  of  the  sugar  remained  in 
the  experimental  cheese.  The  maximum  bacterial  content,  as  meas- 
ured by  the  ordinary  plate  cultures^  was  not  attained  until  after 
the  sixth  week.  The  rate  of  ripening,  as  measured  by  the  change  in 
texture  and  development  of  flavor,  was  correspondingly  slow.  At 
three  months  the  cheese  still  showed  a  spongy  texture  and  scarcely 
any  cheese  flavor. 

THE  PROTECTIVE  ACTION  OF  ACID. 

The  putrefactive  bacteria  of  the  groups  that  are  constantly  present 
in  milk,  and  hence  in  cheese,  are  unable  to  grow  on  account  of  the 
acid  reaction  which  is  maintained  during  the  entire  period  of  ripen- 
ing. This  protective  action  of  acid  was  first  demonstrated  at  the 
Wisconsin  Experiment  Station  by  Babcock  and  Russell,^  who  re- 
moved the  sugar  from  curd  by  washing  in  water.  The  cheese  de- 
veloped most  undesirable  odors  and  tastes,  and  bacteriological  exami- 
nations showed  liquefying  bacteria  to  be  numerous.  If  lactose, 
glucose,  or  cane  sugar  were  added  to  the  washed  curd,  the  ripening 
process  was  much  more  nearly  normal  because  the  acid  reaction  was 
thereby  restored,  due  to  the  fermentation  of  the  added  sugar,  and 
the  putrefactive  organisms  were  inhibited  as  in  a  normal  cheese. 
In  the  experimental  work  on  flavor  development  in  Cheddar  cheese, 
a  number  of  cheeses  were  prepared  from  curd  from  which  the  sugar 
was  removed  by  washing.  In  Plate  I,  figure  2,  a  normal  and  a 
washed-curd  cheese  are  illustrated.  The  washed-curd  cheese  was 
devoid  of  texture,  being  a  soft,  plastic  mass  and  having  no  resem- 
blance to  cheese  in  odor  and  taste.  On  the  right  of  the  picture  is  a 
cheese  made  from  the  same  washed  curd  to  which  acid  had  been 
added,  with  the  result  that  the  firmness  was  restored. 

The  role  of  organisms  of  the  Bacterium  lactis  aoidi  group  in 
Cheddar  cheese  may  be  summarized  as  follows: 

1.  They  favor  the  curdling  process. 

2.  They  favor  the  expulsion  of  the  whey. 

3.  They  permit  of  the  fusing  of  the  curd  particles. 

4.  They  activate  the  pepsin  of  the  rennet  extract. 

6.  They  have  a  protective  action  against  the  putrefactive  bac- 
teria. 
It  is  certain  that  this  group  is  an  essential  factor  in  the  ripening 
of  Cheddar  cheese.    Their  period  of  gro^vth  has  been  believed  to  be 
short,  since  it  has  not  been  supposed  they  can  continue  to  develop 
after  the  disappearance  of  the  sugar.    It  has  been  shown  by  numer- 

1  Babcock,  S.  M.,  Russell,  H.  L.,  Vivian,  A.,  and  Hastings,  E.  G.  Influence  of  sugar  on 
the  nature  of  the  fermentations  occurring  In  milk  and  cheese.  Wisconsin  Agricultural 
Experiment  Station,  Eighteenth  Annual  Report,  pp.  162-176.    Madison,  1901. 


BuL.  150,  BuRCAu  OF  Anjmal  Industry,  U.  S.  Dept.  of  Agriculture 


Plate  I. 


FiQ.  1.— The  Action  of  Rennet  Extract  on  Casein  Suspended  in  Agar  in  the 
Presence  and  in  the  Absence  of  Acid-Forming  Bacteria. 

The  same  number  (902)  was  placed  beneath  both  plates  at  the  time  the  photograph  was  taken. 
The  digestion  of  the  casein  in  the  presence  of  the  acid-forming  bacteria  has  rendered  the 
medium  transparent  and  the  numt)er  apparent,  while  in  the  absence  of  the  acid-forming  bac- 
teria the  dig&stive  action  has  been  almost  nil. 


Fig.  2.— The  Effect  of  Acid  on  Cheese. 

On  the  left  is  a  cheese  made  from  normal  milk  by  the  usual  process;  in  the  center  a  cheese 
made  from  the  same  milk,  the  curd  being  washed  free  of  sugar;  on  the  right  a  cheese  made 
from  the  same  washed  curd,  to  which  acid  had  been  added.  The  washed-curd  cheese  had  no 
bodv  and  was  a  soft,  plastic  mass.    The  addition  of  acid  restored  the  firmness  of  the  body. 


RATE   OF  BACTEEIAL  GROWTH,  ,  21 

ous  investigators  that  the  period  of  maximum  numbers  is  -when  the 
cheese  is  a  few  days  old;  that  thereafter  the  decline  in  numbers  is 
more  or  less  rapid.  In  some  cheeses,  as  will  be  shown  later,  high 
numT)ers  may  persist  for  long  periods  of  time.  It  is  not  usually 
believed  that  they  can  exert  any  marked  action  in  other  lines  than 
those  that  have  been  mentioned  after  growth  ceases,  although  such 
a  belief  may  not  be  well  founded.  Indeed,  some  facts  would  indicate 
otherwise. 

The  ripening  process,  both  in  regard  to  proteolysis  and  develop- 
ment of  jflavor,  continues  long  after  what  is  supposed  to  be  the  period 
of  bacterial  activity.  A  large  amount  of  data  is  available  as  to  the 
proteolytic  changes  going  on  in  the  ripening  cheese,  but  until  recently 
no  data  have  been  available  to  show  that  the  compounds  containing 
no  nitrogen  were  likewise  constantly  changing.  The'  content  of  the 
cheese  in  volatile  fatty  acids  is  constantly  changing,  and  especially 
the  relative  amounts  of  the  different  acids.  To  explain  these  changes 
recourse  must  be  had  to  enzyms,  and  most  probably  to  those  elabo- 
rated by  the  lactic  organisms  or  to  other  types  of  bacteria  that  de- 
velop subsequently  to  what  has  been  called  the  period  of  bacterial 
activity. 

The  data  to  be  presented  will  show  that  Ihe  period  of  activity  is 
not  to  be  measured  by  determining  the  ordinary  types  of  bacteria. 
The  demonstration  of  continued  bacterial  growth  does  not,  of  course, 
eliminate  the  action  of  enzyms  formed  by  the  Bacterium  IocUg  aeidi 
group  of  organisms  that  during  the  early  life  of  the  cheese  make  up 
most  of  the  flora. 

THE  RATE  OF  BACTERIAL  GROWTH   IN   CHEESE. 

In  the  course  of  the  cooperative  work  carried  on  by  the  Dairy 
Division  of  the  Department  of  Agriculture  and  the  Wisconsin  Ex- 
periment Station  a  large  number  of  cheeses  have  been  examined 
bacteriologically.  Some  were  examined  at  frequent  intervals  during 
the  making  process  and  during  the  first  portion  of  the  ripening 
period;  others  were  examined  at  less  frequent  intervals  during  the 
entire  period  of  ripening. 

METHODS  OF  EXAMINATION. 

The  method  of  examination  used  was  the  grinding  with  sterile 
quartz  sand  of  a  sample  of  the  cheese  taken  from  various  parts  of  a 
plug  and  handled  under  aseptic  conditions.  Inoculations  in  vary- 
ing dilutions  were  made  from  the  cheese  suspension  into  lactose-agar 
plates.  At  first  the  period  of  incubation  of  the  plates  was  48  hours 
at  37°  C.  Later  the  incubation  at  37°  was  supplemented  by  a  further 
incubation  at  20°.    This  method  of  incubation  allows  the  develop- 


22  BACTERIOLOGY   OF   CHEDDAR   CHEESE. 

ment  of  the  maximum  nmnber  of  organisms  and  insures  the  growth 
of  the  colonies  to  a  maximum  size,  an  important  point  when  it  is 
desired  to  differentiate  the  various  types  present.  Gelatin  containing 
no  added  sugar  was  used  in  a  portion  of  the  work;  the  plate  cultures 
were  then  incubated  at  18°  to  22°  C.  for  10  days  and  for  even  longer 
periods  when  slightly  lower  temperatures  were  employed. 

RESULTS  OF  THE  WORK. 

The  data  collected  by  means  of  the  methods  that  have  been  used  by 
other  investigators  of  Cheddar  cheese  serve  simply  to  confirm  their 
work  and  to  emphasize  the  number  of  organisms  of  the  Bacterium, 
lactis  acidi  group  that  are  to  be  found  during  the  early  part  of  the 
ripening  period.  A  portion  of  the  data  is  presented  in  Tables  9,  10, 
and  11  because  it  gives  a  very  complete  picture  of  the  bacterial  de- 
velopment during  and  subsequent  to  the  making  of  the  cheese. 

The  maximum  number  of  bacteria  as  measured  by  the  lactose-agar 
plate  cultures  occurs  early  in  the  ripening  process.  In  8  of  the  11 
cheeses  examined,  as  shown  in  Tables  9  and  10,  the  greatest  number 
of  bacteria  was  found  within  48  hours.  In  the  case  of  the  examina- 
tions detailed  in  Table  11,  it  will  be  noted  that  the  maximum  numbers 
of  bacteria  as  determined  by  lactose-agar  plates  have  been  found 
much  later,  namely,  at  the  end  of  the  fourteenth,  forty-fifth,  and 
seventy-seventh  days.  Thus,  in  no  case  was  the  maximum  number 
found  at  the  first  determination.  It  is  not  believed,  however,  that 
these  cheeses,  selected  especially  to  illustrate  certain  points  to  be  dis- 
cussed later,  give  a  true  picture  of  the  initial  development  of  bacteria. 
In  the  case  of  9  of  the  13  to  be  mentioned  later,  the  maximum  number 
of  bacteria  was  found  on  the  first,  second,  or  third  examinations. 
Harding  and  Prucha  ^  obtained  similar  results.  Seven  out  of  9 
cheeses  examined  showed  the  maximum  number  of  bacteria  at  the 
first  examination,  1  at  the  second,  and  1  at  the  fourth. 

It  is  impossible  to  determine  the  time  at  which  the  growth  of  any 
particular  type  of  organism  in  cheese  ceases.  As  the  fermentation 
in  the  cheese  progresses  and  the  accumulation  of  by-products  in- 
creases, cell  death  begins  to  occur.  As  long  as  the  process  of  cell 
division  is  more  rapid  than  death  of  the  cells,  an  increase  in  living 
bacteria  will  be  shown  by  the  plate  cultures.  Soon  death  of  the  cells 
is  more  rapid  than  cell  division.  At  this  point  the  decrease  in  ap- 
parent numbers  begins,  although  growth  may  continue  for  a  much 
longer  period.  It  seems  probable  that  the  growth  of  the  bacteria  of 
the  Bacterium  lactis  acidi  group  continues  until  the  sugar  is  com- 
pletely fermented.    In  milk  the  cessation  of  growth  is  due  to  the 

1  Harding,  H.  A.,  and  Prucha,  M.  J.  The  bacterial  flora  of  Cheddar  cheese.  New 
York  Agricultural  Experiment  Station,  Technical  Bulletin  8.     Geneva,  Dec.,  1908. 


RATE   OF  BACTEEIAL  GEOWTH. 


23 


appearance  of  free  acid,  in  cheese  to  the  disappearance  of  an  essential 
food,  a  fermentable  carbohydrate. 

The  large  numbers  of  bacteria  found  during  the  first  days  are 
striking;  the  number  as  given  in  the  tables  is  far  below  the  actual 
number,  due  to  the  impossibility  of  breaking  up  the  colonies  in  the 
tough  cheese.  It  is  probable  that  the  germ  content  of  cheese  often 
amounts  to  hundreds  of  billions  of  living  bacteria  in  each  gram  of 
the  moist  cheese.  From  what  is  known  of  the  number  of  cells  in  a 
gram  of  moist  bacterial  growth,  it  is  certain  that  at  the  time  the 
maximum  numbers  of  the  organisms  of  the  Bacterium  lactis  acidi 
group  are  found,  at  least  0.1  per  cent  of  the  moist  cheese  consists  of 
bacteria. 

Table  9. — Numbers  of  bacteria  per  gram  in  Cheddar  cheese  as  determined  bp 
lactose-agar  plate  cultures. 

[Numbers  expressed  In  millions.] 


Cheese 
No. 

Days. 

0 

1 

2 

3 

4 

5 

6 

7 

9 

10 

12 

434 
438 
442 
474 
475 
476 
477 

31 
4.5 
1.3 
58 
158 
138 
53 

490 
"133' 
"987' 

""i34" 

637 
292 

489 
3 

21 

43    

1    

""m 

1,657 
1,538 
1,311 
1,925 

i74 

831 

987 

1,344 

2,518 

2,171 

221 



325 

407 
645 
195 
221 

239 
69 

293 
65 

141 

736 

728 

598 

eie 

1,916 

Table  10. — Numbers  of  bacteria  per  gram  in  Cheddar  cheese  as  determined  by 
lactose-agar  plate  cultures. 

[Numbers  expressed  in  millions.] 


Cheese 
No. 

Milk. 

Curd 

at 
salt- 
ing 
time. 

12 
hours. 

Days. 

1 

2 

4 

6 

14 

21 

28 

35 

49 

70 

98 

580 
681 
582 
583 

8 
0.5 

.7 
.5 

160 
326 
912 
839 

332 

1,048 

623 

965 

586 
736 
709 
569 

235 
405 
848 
580 

145 

684 

522 

1,025 

165 
184 
853 
184 

51 
211 
369 
401 

284 
290 
348 
319 

285 
453 
814 
144 

104 
261 
326 
504 

132 
228 
436 

661 

128 
291 
193 
168 

114 

212 

45 

55 

24 


BACTERIOLOGY  OF   CHEDDAE   CHEESE. 


Table  11. — Numbers  of  bacteria  per  gram  in  Cheddar  cheese  as  determined  by 

plate  cultures. 

[Numbers  expressed  in  millions.] 


Cheese  No.  1. 

Cheese  No.  54. 

Cheese  No.  91. 

Cheese  No.  92. 

Number  of  days. 

Lactose- 

agar 
culture. 

Gelatin 
culture. 

Lactose-     ^^^^^^ 
culture.     <^'^'- 

Lactose- 

agar 
culture. 

Gelatin 
culture. 

Lactose- 

agar 
culture. 

Gelatin 
culture. 

2 

150 

250 

150 

4           

3 

8 

61 

120 

400 

340 

11        

8 

14 

14 

32 

1,400 
58 

5.30 
1,600 

500 
35 

22                

2.5 
2 

10 
10 

30 

is     j        70 

37 

270 
1,400 
1,300 

480 

100 

45 

40 

25 

340 

225 

56 

32      1          5i 
51      1          40 

250 

225 

77  

■"'■■ 

86 

36.6 

24 

128 

74 
145 

50 

18 

15 

17.5 

320 
65 

142 

28.5 

23.5 

10.5 

5.5 

250 
100 
132 
145 
38.5 

84 

100      

8.5 

12 

120 

113 

5 

5 

87 

124 

14 

22.5 

12 
12.5 

96 

143 13 

155  1        .   .. 

13 

38 
32 

165 1 

63 
9 
12.5 

176 1           3.5 

2.5 

1  

5.5 

187 

6 

7.5 

2 

1 

According  to  the  determinations  of  MacNeal,  Latzer,  and  Kerr,* 
there  are  about  5,300  billion  colon  bacilli  in  a  gram  of  dry  growth. 
Figuring  on  a  moisture  content  of  90  per  cent,  there  would  be  630 
billion  in  a  gram  of  the  moist  growth.  From  determinations  made 
by  weighing  out  1  gram  of  the  moist  growth  of  a  coccus  form  from 
cheese,  making  a  uniform  suspension  of  this  in  a  known  volume  of 
water,  and  taking  an  equal  volume  of  this  suspension  and  of  normal 
blood  and  counting  the  number  of  bacteria  and  red  corpuscles  we 
have  obtained  an  average  figure  of  1,150  billion  cells  per  gram  of  the 
moist  growth.  The  basis  for  this  method  of  counting  is  the  fact  that 
from  a  known  number  of  red  blood  cells  one  can  figure  the  volume 
used,  since  the  red  cells  are  practically  a  constant  quantity.  The 
average  volume  of  the  colon  organism  is  1.13  cubic  microns;  of  the 
coccus  0.5236.    The  agreement  in  the  determinations  is  thus  very  close. 

It  is  not  to  be  supposed  that,  so  far  as  the  number  of  bacteria  is 
concerned,  all  cheese  will  be  similar.  The  results  shown  in  Table  11 
emphasize  this  point ;  two  of  the  cheeses,  Nos.  1  and  54,  showed  at  no 
time  over  a  few  million  bacteria  per  gram,  while  the  remaining  two 
had  a  consistently  high  germ  content.  Such  differences  may  be  due 
to  many  causes.  The  period  of  maximum  numbers  is  followed  by  a 
decline,  rapid  in  some  instances,  slow  in  others.  At  the  time  the 
cheese  is  fully  ripe  millions  of  living  lactic  bacteria  are  usually  pres- 

1  MacNeal,  Ward  J. ;  Latzer,  Lenore  L. ;  and  Kerr,  Josephine  E.  The  fecal  bacteria  of 
healthy  men.  Journal  of  Infectious  Diseases,  vol.  6,  No.  2,  pp.  123-169,  Apr.  1;  No.  5, 
pp.  571-600,  No'y.  26.     Chicago,  1909. 


THE  ENZYMIC  ACTION  OF   LACTIC  BACTERIA.  25 

ent  in  each  gram  of  cheese.    Living  lactic  organisms  have  been  found 
by  one  of  the  authors  in  a  cheese  over  4  years  old. 

The  ripening  of  the  cheese,  both  in  reference  to  proteolysis  and 
flavor  development,  continues  long  after  this  group  of  organisms  has 
ceased  to  grow.  A  large  amount  of  data  is  available  to  show  that  a 
constant  change  in  the  nitrogenous  bodies  present  is  taking  place.  It 
has  also  been  shown  by  the  authors  that  the  content  of  the  cheese  in 
fixed  and  volatile  acids  changes  during  the  ripening  process.  To 
explain  these  latter  changes,  recourse  must  be  had  to  enzyms  elab- 
orated by  the  lactic  bacteria,  or  else  it  must  be  supposed  that  other 
groups  of  organisms  develop  subsequently. 

THE  ENZYMIC  ACTION  OF  LACTIC  BACTERIA. 

The  work  of  Buchner,  Herzog,  and  others  has  shown  the  presence 
of  an  enzym  in  certain  lactic-acid-producing  bacteria  that  are  essen- 
tially different  from  those  predominating  in  cheese.  This  intracellu- 
lar enzym,  which  can  be  demonstrated  only  after  the  disintegration 
of  the  cell,  forms  small  quantities  of  lactic  acid  from  sugar.  So  far 
as  is  known  to  us,  a  similar  enzym  has  never  been  demonstrated  in 
organisms  of  the  Bacterium  lactis  acidi  group.  The  growth  of  these 
organisms  on  all  media  is  so  meager  that  it  is  very  difficult  to  obtain 
a  sufficient  amount  of  the  growth  so  that  it  can  be  treated  by  methods 
similar  to  those  employed  by  Buchner  and  others.  An  acid-produc- 
ing enzym  in  the  lactic  bacteria  has,  however,  been  demonstrated  by 
quite  different  methods.  It  had  been  noted  that  when  a  sample  of 
raw  or  sterilized  milk  in  which  varying  numbers  of  lactic  bacteria  had 
been  allowed  to  develop,  and  to  which  a  preservative,  as  chloroform 
or  toluol,  had  been  added,  the  cells  soon  disappeared,  or  at  least  could 
no  longer  be  detected  by  microscopical  examination.  It  was  thought 
that  if  the  cells  would  undergo  disintegration  after  having  been 
killed  by  an  antiseptic  while  in  an  actively  growing  condition,  any 
enzyms  present  should  exert  their  peculiar  action  as  well  as  though 
the  cells  were  mechanically  ruptured. 

The  following  experiment  was  planned :  Bottles  of  fresh  raw  milk 
and  of  the  same  milk  heated  to  97°  C.  for  a  short  time  were  in- 
oculated with  a  pure  culture  of  Bacterium,  lactis  acidi.  At  varying 
periods  in  the  development  of  acid  a  portion  of  the  milk  was  re- 
moved and  preserved  with  3  per  cent  of  toluol.  In  the  bottles  treated 
soon  after  inoculation  a  small  number  of  bacteria  were  present,  while 
in  those  to  which  the  preservative  was  added  at  a  later  stage  in  the 
development  of  acid  a  much  larger  amount  of  bacterial  growth  was 
present.  If  any  enzymic  action  occurred,  the  bottles  should  show  a 
quantitative  difference  in  the  amount  of  acid  formed  corresponding 
to  the  amount  of  bacterial  cells  present.  Kaw  milk,  when  preserved 
50978°— Bull.  150—12 i 


26  BACTERIOLOGY   OF   CHEDDAR   CHEESE. 

with  chloroform  or  toluol,  gradually  increases  in  acidity.  This  in- 
crease occurs  when  the  milk  contains  only  the  bacteria  that  come 
from  the  interior  of  the  udder  and  to  which  the  preservative  has  been 
added  as  soon  as  drawn.  In  a  bottle  of  milk  put  up  in  1898  by  Dr. 
S.  M.  Babcock  and  preserved  with  an  excess  of  chloroform  an  acidity 
of  0.7  per  cent  was  found  in  1910,  while  the  sugar  content  was  as 
great  as  iii  the  fresh  milk,  being  5  per  cent.  This  increase  of  acid  has 
been  shown  in  all  samples  of  milk  preserved  by  the  authors.  It  is 
undoubtedly  due  to  the  formation  of  amino  acids  by  the  inherent 
proteolytic  enzyms  of  the  milk. 

In  the  bottles  of  raw  milk  in  the  experiment  two  acid-forming 
factors  might  be  present :  First,  one  forming  an  acid  from  the  pro- 
tein ;  second,  the  enzym  of  the  lactic  bacteria  acting  on  the  sugar.  In 
the  bottles  of  heated  milk  only  the  latter  could  be  active,  since  the 
degree  of  heat  was  sufficient  to  destroy  all  the  inherent  enzyms  of 
the  milk.  The  milks  thus  differently  treated  should  show  a  quantita- 
tive difference  in  increase  in  acid  if  the  bacterial  enzyms  were  capable 
of  such  action.  The  first  bottle  of  the  raw-milk  series  contained  a 
minimum  number  of  bacteria,  since  the  milk  was  but  a  few  hours  old 
and  drawn  under  clean  conditions.  The  acidity  in  this  bottle  was 
that  of  the  fresh  milk.  Three  per  cent  of  a  pure  lactic  organism  in 
milk  was  then  added  to  the  remainder  of  the  raw  milk.  The  second 
bottle  was  filled  immediately  after  the  addition  of  the  culture.  The 
milk  was  then  incubated  and  at  varying  intervals  bottles  were  filled 
and  toluol  added.  The  first  acid  determinations  were  made  at  once 
after  adding  the  toluol  to  each  bottle.  It  will  be  noted  in  Table  13 
that  the  acidity  in  the  case  of  the  raw  milk  varied  from  0.184  to  0.408 
per  cent.  The  increasing  acidity  indicated  a  great  increase  in  the 
numbers  of  bacteria,  which  was  also  shown  by  microscopical  prepa- 
rations made  from  each  bottle  at  the  time  the  preservative  was  added. 

The  set  of  bottles  filled  with  heated  milk  were  treated  in  the  same 
manner  as  the  raw  milk.  The  first  bottle  of  this  set  contained  prac- 
tically no  living  bacteria.  The  acidity  in  the  case  of  the  remainder 
ranged  from  0.203  to  0.456  per  cent. 

In  order  to  determine  how  long  the  cells  persisted  in  an  active  con- 
dition, tubes  of  milk  were  heavily  inoculated  from  the  various  bottles. 
The  results  are  given  in  Table  12. 


BESULTS  OF  INOCULATION   OF   STERILE   MILK. 


27 


Table  32. — Results  of  inoculations  of  sterile  milk  from  the  bottles  of  milk  con- 
taining 8  per  cent  toluol. 


Bottle 

No. 

Tube. 

Growth  from  inoculations 
made  after— 

Iday. 

3  days. 

5  days. 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

1 
2 
1 

2 
1 
2 
1 
2 
1 
2 
1 
2 
1 
2 
1 
2 
1 
2 
1 
2 
1 
2 
1 
2 
1 
2 
1 
2 

+ 
+ 

+ 
+ 
+ 

+ 

+ 
+ 

+ 

- 

The  results  of  the  inoculation  of  sterile  milk  show  that  no  growth 
could  have  taken  place  in  the  bottles  after  the  addition  of  the  toluol, 
and  that  within  a  short  time  the  lactic  organisms  were  all  destroyed. 
Any  acid  formed  in  the  raw  milk  after  the  fifth  day  must  have  been 
due  in  part,  at  least,  to  the  inherent  proteolytic  enzyms  of  the  milk ; 
any  increase  of  acid  in  the  heated  milk  must  have  been  due  to  the 
enzyms  set  free  by  the  disintegrating  cells. 

Microscopical  preparations  wore  made  at  intervals  from  the  various 
bottles.  The  smears  were  stained  with  a  saturated  aqueous  solution 
of  methylene  blue.  'W'^ith  this  stain  the  cells  can  be  distinguished 
for  some  time  after  they  can  no  longer  be  demonstrated  by  the  Gram- 
Weigert  stain,  which  has  been  used  in  the  examination  of  smears 
direct  from  cheese  (See  p.  32.)  In  raw  milk  the  cells  were  agglu- 
tinated into  large  clumps.  The  number  in  the  preparations  made 
at  various  intervals  became  less  and  less,  and  after  45  days  they  had 
completely  disappeared.  As  the  cells  deteriorated  they  stained  more 
and  more  faintly. 

In  the  heated  milk  the  disappearance  of  the  cells  was  less  rapid. 
After  74  days  some  could  be  distinguished,  although  their  outlines 
seemed  to  be  more  or  less  distorted. 

In  the  bottles  the  following  conditions  were  present :  The  mass  of 
cells  varied  widely;  cell  groM^th  undoubtedly  ceased  as  soon  as  the 
antiseptic  was  added  and  within  a  few  hours  the  cells  were  all  de- 
stroyed, having  been  killed  while  in  an  active  condition  by  an  agent 
that  is  supposed  to  have  the  minimum  eflPect  on  the  intracellular 


28 


BACTERIOLOGY   OF   CHEDDAR   CHEESE. 


enzyms.  The  cells  disintegrated  more  or  less  rapidly.  It  would 
seem  that  if  any  acid- forming  enzyms  were  present  in  the  bacterial 
cells  they  should  manifest  themselves  under  these  conditions  by  the 
formation  of  acid.  Acid  determinations  were  made  with  the  greatest 
precautions  possible  under  the  conditions  of  the  experiment.  The 
results  are  given  in  detail  in  Table  13 : 

Tabie  13. — Increase  of  acidity  iti  milk  preserved  with  S  per  cent  toluol. 

RAW  MILK. 


Sample. 


When 
preserv- 
ative 
was 

added. 


Days 


28 


59 


121 


Bottle  No.  1: 

Acidity.. 

Increase.. 
Bottle  No.  2: 

Acidity.. 

Increase.. 
Bottle  No.  3: 

Acidity.. 

Increase.. 
Bottle  No.  4: 

Acidity.. 

Increase- 
Bottle  No.  6: 

Acidity.. 

Increase- 
Bottle  No.  6: 

Acidity.. 

Increase.. 
Bottle  No.  7: 

Acidity.. 

Increase.. 


Per  cent. 
0.184 


.204 
.223 


.266 
.282 
.327 
.408 


Per  cent. 
0.19 
.006 

.22 
.016 

.261 
.038 

.261 
-.005 


Per  cent. 
0.234 
.05 

.257 
.053 

.308 
.085 

.326 
.06 


Per  cent. 
0.273 


.299 
.095 


.332 
.05 


.356 
.029 


.489 
.081 


.404 
.122 


.408 
.081 


.561 
.153 


.377 
.154 


.345 
.079 


.45 
.168 


.443 
.116 


.579 
.171 


Percent. 
0.29 
.106 

.326 
.122 

.359 
.136 

.391 
.125 


Per  cent. 

0.317 

.133 

.349 
.145 

.4 
.177 

.414 
.148 


Per  cent. 

0.368 

.184 

.414 
.21 

.515 
.292 

.492 
.226 


Per  cent. 

0.368 

.184 

.414 
.21 

.506 
.283 

.494 
.228 


.492 
.21 


.515 
.188 


.616 
.208 


.423 
.141 


.538 
.211 


.63 

.222 


.644 
.362 


.644 
.317 


.745 
.337 


.584 
.302 


.676 
.349 


.823 
.415 


HEATED  MILK. 


Bottle  No.  8: 

Acidity... 

Increase... 
Bottle  No.  9: 

Acidity... 

Increase... 
Bottle  No.  10: 

.\cidity... 

Increase... 
Bottle  No.  11: 

Acidity... 

Increase... 
Bottle  No.  12: 

Acidity... 

Increase... 
Bottle  No.  13: 

Acidity... 

Increase... 
Bottle  No.  14: 

Acidity... 

Increase... 


0.184 


.203 
.256 


.308 
.349 
.41 
.456 


0.18 
-.004 


.206 
.003 


.285 
.029 


.356 
.048 


.427 
.078 


.46 
.005 


.494 
.038 


0.175 
-.009 


.202 
-.001 


.317 
.061 


.368 
.06 


.423 
.074 


.519 
.063 


0.197 
.013 


.225 
.022 


.331 
.075 


.395 
.087 


.46 
.111 


.552 
.096 


0.212 
.028 


.258 
.055 


.338 
.082 


.394 


.428 
.079 


.497 
.087 


.528 
.072 


0.243 
.059 


.27 
.067 


.354 


.423 
.115 


.492 
.143 


.515 
.105 


.599 
.143 


0.262 
.078 


.276 
.073 


.363 
.107 


.432 
.124 


.54 
.191 


.561 
.151 


.68 
.224 


0.267 
.083 


.276 
.073 


.372 
.116 


.451 
.143 


.543 
.194 


.672 
.262 


.708 
.252 


From  the  conditions  of  the  experiment  it  is  to  be  expected  that 
the  increase  of  acidity  in  the  raw  milk  would  be  greater  than  in  the 
heated  milk,  and  that  if  the  bacterial  enzyms  were  operative  the 
increase  in  acid  would  be  directly  proportional  to  the  amount  of 
enzym  present  or  to  the  mass  of  cells.  It  will  be  noted  that  these 
are  the  conditions  shown  to  be  present  by  the  figures  given  in  the 
table.  The  data  from  three  bottles  of  each  of  the  raw  and  heated 
milks  are  given  in  graphical  form  in  figures  1  and  2.    The  bottles 


INCREASE  OF  ACIDITY  IN   MILK. 


29 


PE 
.39 

.36 

.33 

.30 
.XT 

.a* 

R  CENT 
AOIOITI 

3F 

// 

^ 

RAW  nil 

K  3PCf 

CENT  ■ 

■QLXJOL 

/ 

^ 

y 

V 

/ 

^ 



,^ 

.31 

A^ 

.15 
.12 
.09 

.03 

/ 

._- 

^ 

y^ 

/ 

-"= 

/ 

y 

fr       1 

5    la   2 

k     30    3 

h    A1,    A 

\    54    6 

3     66    T 

5    76   e 

I 

■V    90     9 

>    loz   1!  a  iM    1 

0  126  i:ia  13a  1-4   "30 

Fig.  1. — Increase  of  acidity  in  raw  milk  preserved  with  3  per  cent  toluol.  Bottle  1  con- 
tained the  minimum  number  of  bacteria,  bottle  7  the  maximum,  and  bottle  4  an 
intermediate  number. 


Pe 

(T   CENT 

SF 

#36 

ACIDITY 

H6r/i 

TEO  n/t 

K  ypcF 

CCNT 

-OLUOL 

Si 
JO 

J6T 

_— 

_—- ^ — 

w|l 

w^^ 

M 

^ 

■' 

^ 

^ 

.13 

...^ 

., .—' 

— 

.*S 

..^ 

.<^^ 

..— -- 

— 

_-— - 

___o___ 

r 

-~p:^ 

_^^^,-— 

.03 

■ 

e     1 

s     la   : 

-*     30    2 

f>    Af,    * 

a  34   « 

3     66    T 

«    TO    s 

( 

4.    SO      < 

W3 

6 

loi;  1 

sa  iM-   1 

so    126    1 

}a  i3a  1 

>4   laa 

Fig.  2. — The  increase  of  acidity  in  heated  mllli  preserved  with  3  per  cent  toluol.  Bottle  8 
contained  the  minimum  number  of  bacteria,  bottle  14  the  maximum,  and  bottle  11  an 
intermediate  number. 


30 


BACTERIOLOGY   OF   CHEDDAR   CHEESE. 


selected  were  the  ones  containing  the  minimum  and  maximum 
numbers  of  bacteria  and  an  intermediate  bottle  from  each  set.  The 
curves  presented  show  the  results  to  be  entirely  consistent  with  what 
might  be  expected. 

In  order  to  make  the  comparisons  more  easy  there  are  given  in 
Table  14  the  rate  of  daily  increase  for  each  bottle  of  the  two  sets. 
The  difference  between  the  increase  of  acid  in  the  raw  milk  and  the 
heated  milk  should  give  the  increase  in  the  raw  milk  due  to  the 
inherent  enzyms  of  the  milk.  This  increase  should  be  constant,  or 
nearly  so,  since  the  same  amount  of  enzym  was  operative  in  each 
bottle,  although  under  somewhat  different  conditions,  as  regards 
acidity  in  the  different  bottles. 


Table  14. 


-Daily  increase  in  acidity  in  bottles  of  raw  and  heated  milk  preserved 
with  toluol. 


Raw  milk. 

Heated  milk. 

Increase 
(due  to 
enzyms 
of  milk). 

Bottle  1 

Per  cent. 
0.0012 
.0014 
.0018 
.0015 
.0020 
.0023 
.0027 

Bottles 

Per  cent. 
0.0005 
.0005 
.0007 
.0010 
.0013 
.0017 
.0017 

Percent. 
0  0007 

Bottle  2 

Bottle  9 

0009 

Bottle  3 

Bottle  10 

.0011 

Bottle  4 

Bottle  11. 

0005 

Bottle  5 

Bottle  12.  .  . 

0000 

Bottle  6 

Bottle  13 

.0007 

Bottle  7 

Bottle  14 

.0016 

It  will  be  noted  that  the  daily  increase  is  proportional  to  the 
amount  of  bacteria  present,  and  that  the  daily  increase  due  to  the 
inherent  enzyms  of  milk  is  as  constant  as  could  be  expected.  The 
comparison  has  been  made  bottle  for  bottle  in  the  two  series.  This 
introduces  an  error,  since  the  amount  of  bacterial  cells  present  in 
the  compared  bottles  of  raw  and  heated  milk  was  not  always  the 
same.  In  the  case  of  bottles  2  and  9,  which  are  compared,  the  initial 
acidity  was  identical,  while  in  the  case  of  bottles  6  and  13^  which  are 
also  compared,  the  initial  difference  in  acidity  was  0.083  per  cent. 
The  results  seem  to  leave  no  doubt  concerning  the  presence  of  an  acid- 
forming  enzym  in  the  organisms  of  the  Bacterium  lactis  acidi  group 
that  acts  on  the  milk  sugar.  It  might  be  thought  that  the  increase 
in  acidity  was  due  to  the  production  of  amino  acids  by  a  proteolytic 
enzym.  In  this  case  the  soluble  nitrogen  must  be  augmented.  That 
^this  does  not  occur  has  been  shown  by  numerous  investigators. 

The  experiment  was  repeated  in  full  detail  with  identical  results. 
The  increase  in  acid  can  not  be  asserted  to  be  due  to  the  formation  of 
lactic  acid,  since,  of  course,  no  qualitative  tests  could  be  made  in  the 
presence  of  the  lactic  acid  present  in  the  milk  at  the  beginning  of 
the  experiment. 


OTHEK  GROUPS  OF  BACTERIA  IN  CHEDDAR  CHEESE.  31 

It  is  true  that  in  cheese  the  sugar  disappears  within  a  few  days, 
and  the  enzym  which  was  active  under  the  conditions  of  the  experi- 
ment could  therefore  not  be' active  in  cheese.  This  group  of  organ- 
isms has  been  believed  to  be  peculifirly  deficient  in  enzyms.  It  has 
even  been  claimed  that  they  were  the  only  bacteria  devoid  of  cata- 
lase;  no  proteolytic  enzyms  have  been  demonstrated  in  them,  and 
heretofore  none  acting  on  carbohydrates  with  the  formation  of  acid. 
It  is  very  probable  that  various  classes  of  enzyms  are  formed  by  the 
lactic  bacteria.  As  has  been  shown,  a  considerable  part  of  the  mass 
of  the  cheese  consists  of  the  cells  of  these  organisms,  which  slowly 
disintegrate  and  their  intracellular  products  are  set  free.  It  is  not 
at  all  improbable  that  these  products  are  the  casual  agents  of  changes 
that  occur  in  the  cheese,  and  that  the  roles  of  the  lactic  bacteria  are 
not  limited  to  those  previously  mentioned. 

OTHER  GROUPS  OF  BACTERIA  IN  CHEDDAR  CHEESE. 

It  is  difficult  to  conceive  that  the  varied  chemical  changes  that 
occur  during  the  ripening  of  Cheddar  cheese  can  be  due  directly  or 
indirectly  to  the  lactic  bacteria  alone.  It  seems  as  though  other 
biological  factors  must  be  operative,  but,  as  was  previously  stated, 
no  one  has  demonstrated  the  constant  occurrence  in  large  numbers 
of  any  other  group  of  bacteria  than  the  lactic  group  in  Cheddar 
cheese.  With  the  purpose  of  making  a  more  complete  examination 
of  the  cheese  during  the  entire  ripening  period,  the  plate-culture 
work  has  been  supplemented  by  other  methods. 

DETERMINATIONS  BY  MILK-DILUTION  AND  PLATE-CULTURE  METHODS. 

Since  no  solid  medium  seemed  to  promise  better  results  than  the 
standard  media  hitherto  employed,  milk  was  chosen  as  the  medium 
most  likely  to  permit  of  the  growth  of  other  groups  of  bacteria 
possibly  present.  In  a  previous  publication^  data  concerning  the 
distribution  of  a  group  of  rod-shaped  lactic  bacteria  in  milk  and 
other  dairy  products  have  been  given.  Among  these  organisms  are 
included  those  found  in  many  fermented  milks,  the  Bacillus  huh 
garicus  group,  also  the  B.  casei  group,  to  which  the  work  of  Von 
Freudenreich  and  Jensen  has  attracted  attention,  as  well  as  many 
of  the  acidophilous  organisms  found  especially  in  the  alimentary 
tracts  of  animals.  These  organisms  were  found  to  be  constantly 
present  in  milk,  butter,  and  in  all  the  samples  of  Cheddar  cheese 
examined.  No  quantitative  analyses  of  cheese  were  made,  however, 
in  the  work  to  which  reference  has  been  made. 

1  Hastings,  E.  G. ;  Hammer,  B.  W. ;  and  Hoffman,  C.  Studies  on  the  bacterial  and 
leucocyte  content  of  vaWk.  Wisconsin  Agricultural  Experiment  Station,  Research  Bulletin 
6.     Madison.  June,  1909. 


32  BACTERIOLOGY   OF   CHEDDAE   CHEESE. 

Von  Freudenreich's  work  showed  the  constant  presence  in  large 
numbers  of  the  lactic  bacilli  in  Emmental  cheese.  In  the  making 
of  this  cheese  they  are  added  to  the  milk  in  great  numbers  in  the 
natural  whey  rennet  employed^  and  the  high  temperature  of  the 
curd  during  the  pressing  of  the  cheese  favors  their  development. 
These  organisms  find  a  more  favorable  condition  for  growth  in  milk 
than  in  any  of  the  usual  media,  indeed  some  of  the  members  of  the 
group  can  not  be  cultivated  except  in  milk. 

With  the  idea  of  determining  the  number  of  the  organisms  of 
this  group  in  cheese,  inoculations  in  varying  dilutions  were  made 
from  cheese  emulsions  into  flasks  of  sterile  milk,  which  were  pro- 
tected from  evaporation  by  tin-foil  caps  and  incubated  at  37°  C. 
The  dilutions  increased  by  a  ratio  of  10,  thus  flasks  of  milk  in  the 
case  of  a  single  cheese  might  be  inoculated  with  amounts  of  a  che<?se 
emulsion  representing  0.001,  0.0001,  and  0,00001  gram.  The  extent 
to  which  the  dilutions  were  carried  depended  on  the  age  of  the 
cheese.  An  attempt  was  constantly  made  to  carry  the  dilutions  to  a 
point  where  some  of  the  flasks  would  remain  sterile.  The  method 
is  thus  a  quantitative  one  for  bacteria  that  will  grow  under  the 
conditions  obtaining.  The  dilution  method  is  to  be  considered  a 
rough  way  of  determining  the  number  of  certain  classes  of  bacteria. 

After  incubation  for  one  month  at  37°  C.,  the  acidity  of  each  flask 
was  determined.  The  Bacterium  lactis  acidi  group  of  organisms 
produce  in  average  milk  an  acidity  ranging  from  0.7  to  1.25  per  cent. 
The  lactic  bacilli  produce  an  acidity  usually  exceeding  1.25  per  cent. 
It  has  been  the  practice  to  infer  that  every  flask  of  milk  showing  an 
acidity  above  1.25  per  cent  after  one  month's  incubation  contains  the 
lactic  bacilli,  either  in  pure  culture  or  mixed  with  organisms  of  the 
Bacterium  lactis  acidi  group.  In  the  flasks  showing  an  acidity  less 
than  1.25  per  cent  it  can  not  be  inferred  that  the  lactic  bacilli  are 
absent,  since  some  cultures  are  found  that  produce  no  more  acid  than 
Bacterium  lactis  acidi.  Many  microscopical  examinations  were  made 
of  the  flasks  to  establish  the  accuracy  of  the  conclusions  drawn  from 
the  data  obtained  by  titrations.  Microscopic  examinations  were  also 
made  of  all  the  flasks  from  which  the  titrations  did  not  give  con- 
clusive evidence  concerning  the  organisms  present.  The  smears  were 
stained  with  Gram's  stain  and  decolorized  with  a  mixture  of  one  part 
of  anilin  oil  and  two  parts  of  xylol.  This  method  of  decolorizing 
removes  the  stain  from  the  casein,  but  not  from  any  of  the  organisms 
that  have  been  found  in  cheese. 

The  dilution  method  thus  gives  information  concerning  the  ab- 
solute and  relative  number  of  acid-forming  bacteria  of  these  two 
groups.  It  may  also  give  information  concerning  the  presence  of 
still  other  types  of  bacteria  when  they  are  present  in  greater  numbers 
than  those  of  the  groups  already  referred  to,  since  they  wiU  then 
appear  in  the  flasks  in  pure  culture. 


NUMBER   OF   BACTERIA  IN   CHEESE. 


33 


In  Table  15  are  given  the  results  of  the  analysis  of  four  cheeses. 
A  portion  of  the  data  has  been  presented  in  Table  2  and  is  here 
repeated  for  ease  of  comparing  the  numbers  of  baxiteria  as  determined 
by  plate-culture  methods  and  by  the  dilution  method.  It  will  be 
noted  that  the  dilution  method,  as  a  rule,  gives  higher  results  than 
the  plate  culture.  Out  of  49  determinations,  the  dilution  method 
gave  higher  results  than  the  lactose-agar  plate  determinations  in  30 
cases.  It  is  probable  that  the  dilution  method  always  gives  higher 
results.  If  the  results  show  a  growth  in  a  dilution  of  1  to  10 
millions,  while  the  dilution  of  1  to  100  millions  gives  a  negative 
result,  it  can  only  be  asserted  that  the  bacterial  content  of  the 
cheese  was  between  10  and  100  millions.  It  will  be  noted  that 
the  dilution  method  often  shows  many  fold  more  bacteria  than 
the  plate  cultures,  and  that  the  reverse  is  net  often  true.  In 
cases  where  the  latter  gives  the  higher  number,  the  excess  is  not 
great,  only  three  or  four  times  greater.  The  only'  explanation  that 
can  be  given  of  the  higher  numbers  obtained  by  the  dilution  method 
is  that  some  types  of  bacteria  present  in  the  cheese  that  do  not  appear 
on  the  plate  cultures  find  conditions  favorable  for  growth  in  the  milk. 
The  results  obtained  by  the  dilution  method  indicate  that  other 
organisms  than  the  Bacterium  laptis  acidi  group  are  present  in  the 
cheese,  undoubtedly  in  great  numbers. 

Table  15. — Number  of  bacteria  per  gram  of  cheese  as  determined  on  lactose 
agar  and  gelatin  plates,  and  in  flasks  of  milk  inoculated  with  high  dilutions 
of  cheese. 

[Numbers  expressed  in  millions.] 


Age. 

Cheese  No.  1. 

Cheese  No.  54. 

Cheese  No.  91. 

Cheese  No.  92. 

Lac- 
tose 
agar. 

Gela- 
tin. 

Milk 
dilu- 
tion. 

Lac- 
tose 
agar. 

Gela- 
tin. 

Milk 
dilu- 
tion. 

Lactose 
agar. 

Gelatin. 

Milk 
dilution. 

Lac- 
tose 
agar. 

Gela- 
tin. 

Milk 
dilution. 

Days. 

2 

4 

8 

11 

14 

22 

30 

37 

45 

66 

77 

86 

100 

113 

124 

143 

155 

165 

176 

187 

208 

150 

1,000 
10,000 

1,000 

100 

10,000 

1,000 

250 

150 

100 

1,000 

100 

100 

1,000 

10,000 

3 

10 

61 

120 

400 

340 

8 

14 

10 

32 

1,000 

1,400 
58 

530 
1,600 

s66 

35 


2.5 
2 

10 
10 

1,000 
1,000 

15 
32 

70 

10 
100 

270 
1,400 
1,300 

480 

1,000 
100 
100 

1,000 

1,000 

10 

40 

25 

10 

340 
225 

100 
225 

32 
51 

51 
40 

10 
1,000 

250 

36.5 

24 

10 

128 

74 
145 

50 

18 

15 

17.5 

320 
65 

142 

28.5 

23.5 

10.5 

5.5 

100 
100 
100 
100 
100 
10 
100 

250 
100 
132 
145 

38.5 

63 
9 

84 
120 
87 
96 
38 
32 
5.5 

100 

1,000 

1,000 

100 

10 

100 

10 

8.5 

12 

10 

5 

5 

10 

14 
22.5 

12 
12.5 

10 
100 

13 

13 

10 

3.5 

2.5 

1 

6 

1 

1 
100 

7.5 

10 

12.5 

2 

100 

2 

1.5 

10 

From  the  results  obtained  from  the  determinations  of  the  degree 
of  acidity  attained  by  the  flasks  of  milk  inoculated  with  varying 


34 


BACTERIOLOGY  OF   CHEDDAR   CHEESE. 


amounts  of  cheese  emulsion,  and  from  the  microscopic  examinations 
of  the  same  flasks,  it  has  been  possible  to  determine  the  relations 
existing  between  the  different  groups  of  acid-forming  organisms,  as 
well  as  their  absolute  numbers. 

Table  16  has  been  constructed  from  such  data.  For  ease  in  com- 
parison the  same  data  are  expressed  in  Table  17  in  percentages,  and 
two  additional  cheeses  are  also  included  in*  the  latter  table.  It  will 
be  noted  from  the  data  presented  in  Table  17  that  during  the  early 
part  of  the  ripening  period  the  organisms  of  the  Bacterium  lactis 
acidi  group  make  up  over  90  per  cent,  and  in  many  cases  approxi- 
mately 100  per  cent,  of  the  acid-producing  flora  of  the  cheese.  With 
increasing  age  the  ratio  changes,  until  late  in  the  ripening  period 
the  rod-shaped  lactic  bacilli  predominate,  and  in  many  cases  make 
up  over  90  per  cent  of  the  acid-forming  bacteria  found  in  the  cheese. 

It  will  also  be  noted  from  the  data  given  in  Table  15  that  the  period 
at  which  the  maximum  number  of  bacteria  is  found,  as  determined 
by  the  dilution  method  in  milk,  is  coincident  with  the  appearance  of 
the  lactic  bacilli,  as  shown  in  Table  16.  Using  any  culture  medium 
that  furnishes  favorable  conditions  for  the  growth  of  both  groups 
of  lactic  bacteria,  the  maximum  number  of  organisms  should  be 
found  at  the  time  when  the  group  first  to  appear — the  Bacterium 
lactis  acidi  group — has  attained  its  maximum  development,  but  be- 
fore any  considerable  number  of  the  cells  have  died,  and  at  the  time 
when  the  second  group  of  organisms  finds  favorable  conditions  for 
growth  in  the  cheese. 

Table  16. — 'Numbers  of  Bacterium  lactis  acidi  and  of  acid-producing  hacilli  in 
cheese  as  determined  in  milk  cultures. 


[Numbers  expressed  in  millions.] 

Age. 

Cheese  No.  1. 

Cheese  No.  54. 

Cheese  No.  85. 

Cheese  No.  91. 

Cheese  No.  96. 

Bact. 
lactis 
acidi. 

Acid- 
pro- 
ducing 
bacUli. 

Bact. 
lactis 
acidi. 

Acid- 
pro- 
ducing 
bacilli. 

Bact. 
lactis 
acidi. 

Acid- 
pro- 
ducing 
bacilli. 

Bact.  lac- 
tis acidi. 

Acid- 
pro- 
ducing 
bacilU. 

Bact.  lac- 
tis acidi. 

Acid- 
pro- 
ducing 
bacillL 

Days. 

2. 

1,000 
10,000 

1,000 

100 

10,000 

1,000 

■"■"io  ' 

100 

i" 

100 

1,000 
100 

1,000 

100 

10,000 

1.000 

i"" 

1 

4 

8 

10 

1 

1,000 

11.... 

14.... 

10 

1 

1,000 

10 
10 

1,000 

10 

22.... 
30.... 
37.... 

1,000 
.100 

1,000 
100 

10 
10 

■  "io" 

10 
100 

10 

1,000 

100 

10 

1 

100 

10 

1 

10 
10 
10 
10 
10 
1,000 

1,000 
100 
100 

100 
100 
100 

100 
1,000 
100 
100 
100 
100 
100 
100 
100 
10 

10 

1 

10 

1 

10 
10 
10 

1 
1 

10 
10 

45.... 
56.... 

10 

10 

10 
100 

i 

1 

77 

86.... 
100... 

1 

10 

100 
100 
100 
100 
100 
10 
100 

100 
100 
10 
10 
10 
10 
lOO 

10 

1 

10 
10 
10 
....... 

113.   . 

1 

124... 

10 
100 

1 
10 

143... 
155... 

10 

10 

165... 

I 

176... 
187... 

i 

1 

1 
10 

100 
10 

1 

1 

100 

10 

10 

208. 

10 

RESULTS   OF   PLATE   CULTURES. 


35 


Table  17. — Relative  proportion  of  the  Bacterium  lactis  addi  type  and  the  lactic 
acid-producing  hacilU,  as  determined  hy  dilution  cultures  in  milk. 


Age. 

Cheese  No. 
1. 

Cheese  No. 
54. 

Cheese  No. 
58. 

Cheese  No. 
85. 

Cheese  No. 
91. 

Cheese  No. 
92. 

Cheese  No. 
96. 

Bact. 
Uctis 
aculi. 

Acld- 
pro- 
auo- 
ing 
ba- 
culi. 

Bact. 
lactis 
acidi. 

Acid- 
pro- 
duc- 
ing 
ba- 
cUU. 

Bact. 
lactis 
acidi. 

Acid- 
pro- 
duc- 
ing 
ba- 
cilli. 

Bact. 
lactis 
aciai. 

Acid- 
pro- 
duc- 
ing 
ba- 
cUU. 

Bact. 
lactis 
addi. 

Acid- 

pro- 

duo- 

ing 

ba- 
cillL 

Bact. 
lactis 
acidi. 

Acid- 
pro- 
duo- 
ing 
ba- 
cUU. 

Bact. 
lactis 
acidi. 

Acid- 
pro- 
duc- 
ing 
ba- 
cilli. 

Days. 

8 

11 

14 

22 

30 

37 

45 

56 

77 

86 

100 

113 

124 

143 

155 

165 

176 

187 

208 

P.ct. 
90.9 

P.ct. 
9.1 

P.ct. 

P.ct. 

P.ct. 

P.ct. 

P.ct. 

P.ct. 

P.ct. 

99.9 
90.9 

P.ct. 

0.1 

9.1 

P.ct. 

99.9 

99 

99.9 

99.9 

99.9 

P.ct. 
0.1 

1 

P.ct. 

P.ct. 

90.9 

9.1 

99 

1 

99 

1 

90.9 

9.1 

99.99 
90.9 

.01 
9.1 

99.99 
99.9 

0.01 

.1 

60 
50 

50 
60 

9.1 
50 
99 

9.1 
99 
99 
60 
60 
50 
50 

1 

90.9 
60 

1 
90.9 

1 

1 
50 
60 
50 
50 
99 

99 
50 

1 
60 

90.9 

50 

50 

9.1 
50 
60 

90.9 

90.9 

50 

90.9 

60 

90.9 

50 

90.9 

50 

90.9 

50 

9.1 

9.1 
50 

9.1 
50 

9.1 
50 

9.1 
50 

9.1 
SO 

90.9 
99.9 
90.9 
99 
90.9 
90.6 
90.9 
99 
99 
50 
1 

9.1 
.1 

9.1 

1 

9.1 

9.1 

9.1 

1 

1 

50 
99 

60 

60 

90.9 
99 

9.1 
1 

50 
60 

60 
60 

9.1 

90.9 

60 

60 

90.9 

90.9 

90.9 

60 

50 

60 

50 
9.1 
9.1 
9.1 

60 

50 

90.9 

9.1 

90.9 
90.9 

9.1 
9.1 

9.1 
9.1 

90.9 
90.9 

60 

60 

1 

99 

1 

99 

1 

1 

50 

99 
99 
60 

60 

60 

50 
.1 

50 
99.9 

99 
1 

■99'" 

1 

99 

1 

99 

1 

99 

i 

Since  some  of  the  lactic  baicilli  develop  on  lactose  agar  plates,  it 
was  thought  that  if  all  of  the  colonies  on  a  certain  portion  of  each 
plate  were  inoculated  into  milk  one  should  obtain  information  con- 
cerning the  sequence  of  development  of  the  different  groups  of  lactic 
bacteria  in  the  cheese  and  also  concerning  the  ratio  existing  between 
them. 

For  this  purpose  an  area  showing  well-isolated  colonies  has  been 
circumscribed,  and  every  colony  within  the  area  has  been  inoculated 
into  milk.  It  has  been  inferred  that  all  tubes  that  curdled  within 
48  hours  at  37°  C.  contained  Bacterium  lactis  addi.,  while  those  that 
curdled  between  the  second  and  tenth  day  contained  the  lactic  bacilli. 
Enough  control  work  was  done  to  show  that  this  method  of  differen- 
tiation is  sufficiently  accurate  for  the  purpose  in  hand.  The  tubes 
that  did  not  curdle  in  10  days  were  examined  microscopically  to 
determine  the  organism  present. 

Six  cheeses  have  been  thus  examined  at  frequent  intervals  during 
the  period  of  ripening.  The  data  are  given  in  Table  18.  It  will  be 
noted  that  the  results  are  confirmatory^  of  those  obtained  by  the  dilu- 
tion method  in  milk,  although  not  so  striking,  since  many  of  the 
lactic  bacilli  do  not  develop  on  the  plate  cultures.  The  period  at 
which  the  lactic  bacilli  appear  is  later  than  in  the  previous  exami- 
nations (Table  16),  which  demonstrated  their  presence  in  consider- 
able numbers  within  the  first  week  of  the  ripening  period.  The  pre- 
vious examinations  were  made  with  dilution  cultures  in  milk.    In 


36 


BACTEKIOLOGY   OF   CHEDDAR   CHEESE. 


such  the  lactic  bacilli  may  make  themselves  evident  when  they  are 
present  in  very  small  numbers  as  compared  with  the  Bacterium  lactis 
acidi.  With  the  plate-culture  method,  unless  they  are  present  in 
considerable  numbers,  their  presence  in  the  cheese  is  not  likely  to  be 
detected.  This  error  in  the  method  has  been  previously  pointed  out. 
In  the  plate  cultures  they  are  likely  to  be  missed  unless  they  are  pres- 
ent in  a  ratio  of  1  to  10  of  Bacterium  lactis  acidi. 

The  same  cheeses  have  also  been  examined  by  the  dilution  method 
in  milk.  The  results  are  given  in  Table  19.  It  will  be  seen  that 
they  are  confirmatory  of  the  previous  analyses. 

The  great  delicacy  of  the  dilution  method  as  a  means  of  detecting 
the  lactic  bacilli  in  the  presence  of  Bacterium  lactis  acidi  is  shown 
in  the  results  given  in  Table  19.  On  the  examination  of  cheese  308C 
at  two  days  the  percentage  of  Bacterium  lactis  acidi  is  given  as 
99.9999  and  that  of  lactic  bacilli  as  0.0001.  By  this  is  meant  that 
Bacterium  lactis  acidi  was  detected  in  dilutions  approximately  one 
million  times  greater  than  the  lactic  bacilli.  For  example,  the  acidity 
of  the  flask  inoculated  with  one  ten-thousandth  gram  of  cheese  was 
1.64  per  cent,  thus  indicating  the  presence  of  lactic  bacilli,  while  the 
flask  inoculated  with  one  ten-billionth  gram  of  the  cheese  had  an 
acidity  of  0.87  per  cent,  indicating  Baxiterium  lactis  acidi. 


Table  18. — Tlie  relative  proportions  of  Bacterium  lactis  acidi  and  lactic  bacilli 
in  cheese  as  determined  by  plating  on  lactose  agar. 


Cheese  No. 
307C. 

Cheese  No. 

308C. 

Cheese  No. 
309C. 

Cheese  No. 
310C. 

Cheese  No. 
311C. 

Cheese  No. 
312C. 

Age. 

Bact. 
lactis 
acidi. 

Lactic 
bacilli. 

Bact. 
lactis 
ttci^. 

Lactic 
bacilli. 

Bact. 
lactis 
acidi. 

Lactic 
bacilli. 

Bact. 
lactis 
acidi. 

Lactic 
bacLll. 

Bact. 
lactis 
acidi. 

Lactic 
bacilli. 

Bact. 
lactis 
acidi. 

Lactic 
bacilli. 

Milk  before  ren- 
net was  adaed 

When  put  in 
press 

P.ct. 
100 

100 

100 
100 
100 
100 
100 

P.ct. 
0 

0 

0 
0 
0 
0 
0 

P.ct. 
100 

100 

100 
100 
100 
86 

P.ct. 
0 

0 

0 
0 
0 
14 

P.ct. 
100 

100 

100 
100 

P.ct. 
0 

0 

0 
0 

P.ct. 
100 

100 

100 

P.ct. 
0 

0 

0 

P.ct. 
100 

100 

100 
100 
100 
100 

P.ct. 
0 

0 

0 
0 
0 
0 

P.ct. 

100 

100 

100 
100 

P.ct. 
0 

0 

When  taken 
from  press.... 

0 
0 

3  days 

4  days... 

90 
100 

10 
0 

100 
100 

0 
0 

6days.... 

100 

100 
86 
90 

100 
80 

100 

0 

0 
14 
10 

0 
20 

0 

100 
100 
100 
100 
100 
100 

0 
0 
0 
0 
0 
0 

100 
100 
100 

90 
100 
100 

70 

0 
0 
0 

10 
0 
0 

30 

100 
100 

0 
0 

100 
100 

6 

0 

9  days 

100 

86 

0 

14  days 

100 
33 

0 

66 

100 
90 

0 
10 

14 

18  days 

21  days 

22  days 

100 

0 

28  days 

100 

0 

86 

14 

90 

10 

0 

100 

33  days 

36  days 

100 
100 
100 
90 
90 

0 
0 
0 
10 
10 

42  days  . . 

89 

11 

49  days 

29 
40 

71 

00 

56  days 

71 

29 

64  days 

RESULTS  OF  PLATE   CULTUEES. 


37 


Table  19. — The  ratio  bettccen  the  nwnbers  of  Bacterium  lactis  acidi  and  the 
lactic  baciUi  at  different  stages  in  the  ripening  of  Cheddar  cheese,  as  deter- 
mined by  milk-dihition  method. 


Cheese  No. 
307C. 

Cheese  No. 
308C. 

Cheese  No. 
309C. 

Cheese  No. 
310C. 

Cheese  No. 
3nc. 

Cheese  No. 
312C. 

Age. 

Bad. 
lactis 
acidi. 

Acid- 
pro- 
duo- 
ing 
ba- 
cilU. 

Bad. 
lactis 
acidi. 

Acid- 
produc- 
ing 
bacilU. 

Bad. 
lactis 
acidi. 

Acld- 
pro- 
duo- 
ing 
ba- 
cilli. 

Bad. 
lactis 
acidi. 

Acld- 
pro- 
duo- 
ing 
ba- 
cilU. 

Bad. 
lactis 
acidi. 

Acld- 
pro- 
duc- 
ing 
ba- 
cilli. 

Bad. 
lactis 
acidi. 

Acld- 
pro- 
duc- 
ing 
ba- 
cilli. 

Milk    before 
rennet  was 
added 

Perct. 

Perct. 

Perct. 

Perct. 

Perd. 
99.99 

99.9 

99.9 
99 

Perd. 
0.01 

l' 

Perd. 

Perct. 

Perd. 
99 

99.99 

Perd. 
1 

.01 

Perd. 
99 

Perd. 
1 

Just     before 
put  in  press 

When  taken 
from  press. . 

2days 

3  days 

4days 

6days 

99.999 

99.99 
99.9999 
99 
99.9 

.001 

.01 
.0001 

1 
.1 

g9.99 

99.99 

99.9 

99.99 

99.99 

0.01 
.01 
.1 
.01 
.01 

99 
99 

1 

99.9 
50 
99 
99.99 

0.1 

50 
1 
,01 

99 

90.9 

99.99 

1 

9.1 
.01 

1 

99.9 

99 

99.9 

99 

50 

50 

90.9 

50 

90.9 

50 

50 

l' 

l' 

50 
50 

9.1 
50 

9.1 
50 
50 

99 

1 

90.9 

90.9 

90.9 

60 

90.9 

50 

99 

90.9 

9.1 
9.1 
9.1 

50 
9.1 

50 
1 
9.1 

99.9 
90.9 

.1 

7days 

9days 

11  days 

14days 

18  days 

21  days 

28  days 

33  days 

90.9 

50 

99 

99 

50 

90.9 

50 

9.1 
50 
1 

1 
50 

9.1 
50 

99.9 
99 

.1 

1 

90.9 
99 

9.1 

1 

9.1 

90.9 

9.1 

90.9 

9.1 

50 

60 

35  days 

42  days 

60 
50 

60 
50 

60 
99 

50 

1 

90.9 

9.1 

It  must  not  be  inferred  that  the  figures  given  in  the  tables  repre- 
senting the  results  obtained  by  the  dilution  method  indicate  the  ex- 
act number  of  the  different  groups  of  lactic  organisms  or  the  exact 
ratio  existing  between  them.  The  sudden  increase  or  decrease  of  the 
ratio  is  due  to  the  inherent  errors  of  the  dilution  method.  It  would 
be  an  endless  task  to  show  by  this  method,  or  any  other,  m  fact,  the 
exact  proportion  between  the  different  types  of  organisms  in  the 
cheese  at  various  stages  in  the  ripening  period. 

From  the  data  presented  there  would  seem  to  remain  no  doubt  that 
the  lactic  bacilli  develop  later  than  the  Bacterium  lactis  acidi  group, 
making  their  appearance  within  a  week  or  10  days  after  the  cheese 
is  made,  gradually  increasing  in  numbers  and  probably  attaining 
their  maximum  during  the  first  month  and  then  gradually  decreasing 
in  numbers.  The  number  found  per  gram  of  the  cheese  ranges  from 
a  few  million  to  one  billion.  Usually  they  do  not  attain  such  great 
numbers  as  do  the  ordinary  lactic  bacteria.  Again,  their  numbers 
may  equal  the  lactic  bacteria,  as  in  cheese  No.  1,  Table  16. 


DETERMINATIONS  BY  MICROSCOPIC  EXAMINATION  OF  THE  CHEESE. 

It  was  also  thought  that  the  gradually  changing  flora  should  mani- 
fest itself  in  the  appearance  of  microscopic  preparations  made  from 
the  cheese.    At  the  various  periods  of  sampling  smear  preparations 


38 


BACTERIOLOGY   OF   CHEDDAR  CHEESE. 


were  made  from  the  emulsions  and  stained  with  Gram-Weigert's 
stain  as  described  on  page  32. 

The  preparations  show  in  a  general  way  the  same  change  in  flora 
as  has  already  been  made  evident  by  the  analyses  presented.  It  is 
not  to  be  expected  that  the  microscopic  examination  would  give  re- 
sults as  striking  as  the  cultural,  since  the  latter  measures  the  living 
cells,  the  former  only  those  cells  that  have  not  lost  their  staining 
properties. 


Z  DAYS 


57  DAYS 


112  DAYS  J  59  DAYS 

Pig.  3. — Typical  microscopic  fields  from  cheese  No.  54  at  different  stages  in  the  ripenin;,' 
process.     T Camera  lucida  drawings.) 

Figures  3  and  4  have  been  prepared  from  camera-lucida  drawings 
of  typical  microscopical  fields  of  several  slides  made  from  2  cheeses. 
The  results  of  the  cultural  examination  have  been  presented  in 
Tables  15  and  16.  Figure  3  shows  that  at  2  and  57  days  the  organ- 
isms are  almost,  if  not  wholly,  of  B.acterium  lactis  acidi  group;  at 
112  days  there  is  a  preponderance  of  Bacterium  lactis  acidi  and  a  few 
lactic  bacilli,  while  at  18J>  days  but  few  Bacterium  lactis  acidi  cells 


RESULTS  OF  MICROSCOPIC  EXAMINATIONS. 


39 


remain,  the  lactic  bacilli  having  become  more  evident.  Figure  4  in- 
dicates likewise  that  at  29  and  78  days  the  organisms  are  wholly  of 
the  Bacterium  lactis  dcidi  group,  while  at  121  and  185  days  a  decrease 
is  seen  in  Bacterium  lactis  acidi  and  an  increase,  in  lactic  bacilli.  It 
will  be  noted  that  rod-shaped  organisms  do  not  appear  nearly  as 
soon  as  would  be  indicated  by  the  cultural  analyses,  but  that  they 
do  at  last  appear,  especially  when  the  cells  of  the  lactic  bacteria  have 
greatly  decreased  in  numbers.    This  may  again  be  taken  as  evidence 


29  DAYS 


7G  DAYS 


\Z\  DAYS  IS5DAY5 

Fig.  4. — Typical  microscopic  fields  from  cheese  No.  91  at  different  periods  In  the  ripening 
process.     (Camera  luclda  drawings.) 

that  the  lactic  bacilli  develop  subsequent  to  the  Bacterium  lactis 
acidi  group  and  that  they  tend  to  disappear  less  rapidly. 

DISAPPEARANCE  OF  BACTERIAL  CELLS  IN  CHEESE. 

The  drawings  presented  in  figures  3  and  4  are  evidence  of  the  grad- 
ual disappearance  and  disintegration  of  the  bacterial  cells  in  cheese. 
It  is  a  well-known  fact  that  the  enzyms  elaborated  by  bacteria  in 
pure  cultures  continue  to  act  long  after  the  death  of  the  cells.    There 


40  BACTERIOLOGY   OF   CHEDDAR   CHEESE. 

are  many  reasons  for  believing  that  there  is  no  enzym  action  until 
cell  disintegration  begins.  It  has  been  previously  shown  that  the 
Bacterium  lactis  acidi  group  of  bacteria  elaborate  enzyms  that  are 
able  to  increase  the  acidity  of  milk.  As  previously  indicated,  it  may 
be  surmised  that  still  other  types  of  enzyms  are  formed  by  this  group 
of  bacteria.  The  direct  influence  of  the  immense  number  of  acid- 
forming  bacteria,  amounting  to  billions  per  gram,  must  be  of  great 
importance  in  cheese  ripening;  the  indirect  action  through  their 
enzyms  may  be  still  greater. 

It  is  certainh'^  true  that  in  normal  Cheddar  cheese  the  period  of 
development  of  the  ordinary  lactic  bacteria — the  organisms  which 
heretofore  have  been  believed  to  be  the  only  ones  of  importance  in 
cheese  because  of  their  constant  presence  in  great  numbers — is  fol- 
lowed by  the  development  of  another  group  of  organisms,  which, 
while  they  ferment  milk  sugar,  producing  lactic  acid,  must  have 
some  other  source  of  carbon  in  cheese  on  account  of  the  total  disap- 
pearance of  the  sugar  before  their  maximum  period  of  development. 
The  influence  of  the  first  group  has  been  pointed  out  early  in  this 
paper.  The  role  of  the  second  gi-oup  can  not  at  this  time  be  de- 
termined, but  because  of  their  constant  presence  in  numbers,  approxi- 
mating if  not  equaling  those  of  the  first  group,  their  influence  on 
the  ripening  of  the  cheese  can  not  be  a  minor  one. 

DETAILED   STUDY   OF  LACTIC  BACILLL 

The  work  of  Von  Freudenreich  and  numerous  later  investigators 
indicates  that  the  lactic  bacilli  represent  a  group  with  certain  com- 
mon characteristics,  such  as  morphology,  optimum  temperature  for 
growth,  and  to  some  extent  in  the  production  of  compounds  from  the 
sugar  fermented.  In  all  fields  of  bacteriology  it  is  difficult  at  the 
present  time  to  draw  the  boundary  lines  of  any  group  of  bacteria. 
The  placing  of  the  lactic  bacilli  found  in  cheese  in  a  single  group 
may  be  incorrect,  but  because  of  the  fact  that  all  the  cultures  studied 
have  certain  characters  in  common,  it  seems  best  to  discuss  them  as 
a  single  group,  although  more  detailed  study  might  divide  them  into 
a  number  of  groups.  A  comparative  study  has  been  made  of  20  pure 
cultures  isolated  in  the  course  of  the  work!  The  main  facts  of  this 
stud}^  are  here  presented.  All  the  cultures  were  isolated  from  cheese 
with  the  exception  of  No.  160,  which  was  obtained  from  milk.  Cul- 
tures 58A  and  58B  represent  different  colonies  from  plates  made  to 
insure  the  purity  of  the  cultures  to  be  used  in  the  detailed  study. 
The  differences  noted  in  the  study  of  these  two  cultures  originally 
from  the  same  culture  illustrate  the  differences  one  may  expect  to 
obtain  in  cultural  work. 

The  action  of  the  different  cultures  in  milk  is  given  in  Table  20,  in 
which  they  have  been  arranged  according  to  the  degree  of  acidity 
produced  in  milk. 


DETAILED  STUDY  OF  LACTIC  BACILLI.  41 

ACIDITY  PRODUCED. 

It  will  be  noted  from  the  data  in  Table  20  that  both  the  rate  of 
acid  development,  and  hence  the  time  required  to  curdle  milk,  as 
well  as  the  maximum  amount  of  acid  produced,  varies  widely,  the 
acidity  varying  from  0.91  per  cent  to  2.31  per  cent.  The  acidity 
produced  by  each  culture  increased  after  the  tenth  day.  Since  the 
flasks  were  protected  by  tin-foil  caps,  this  increase  can  not  have  been 
due  to  evaporation. 

White  and  Avery ,^  in  studying  cultures  from  fermented  milks, 
noted  the  same  differences  in  acid  production.  They  established 
two  groups,  one  producing  an  acidity  in  milk  of  about  1  per  cent, 
the  other  of  3  per  cent,  in  10  days.  Such  a  division  of  the  cultures 
studied  could  not  be  made. 

Rogers  ^  states  that  a  typical  culture  of  the  lactic  bacilli  from 
fermented  milk  produces  nearly  3  per  cent  of  acid  in  three  days  at 
37°  C.  Von  Freudenreich  and  Thoni*  studied  cultures  from  Em- 
mental  cheese  that  produced  from  0.2  to  1.26  per  cent  of  acid  in 
whey  to  which  peptone  had  been  added. 

Out  of  the  several  hundred  titrations  made  of  milk  inoculated 
with  cheese  emulsions  but  15  showed  an  acidity  above  2  per  cent. 
When  raw  milk  is  incubated  at  37°  C,  the  acidity  will  often,  if  not 
in  the  majority  of  cases,  exceed  2  per  cent  and  at  times  may  reach 
3.5  to  4  per  cent.  It  would  thus  seem  that  certain  lactic  bacilli  pres- 
ent in  milk  do  not  find  favorable  conditions  for  development  in 
Cheddar  cheese. 

FORMS  OF  LACTIC  ACID  PRODUCED. 

A  number  of  investigators  have  determined  the  rotary  power  of 
the  lactic  acid  formed  by  the  lactic,  bacilli.  Bertrand  and  Weis- 
weiller*  concluded  that  the  acid  was  a  mixture  of  the  levo  and 
dextro  forms,  with  a  slight  predominance  of  the  latter. 

1  White,  Benjamin,  and  Avery,  Oswald  T.  Observations  on  certain  lactic  acid  bacteria 
of  the  so-called  Bulgaricus  type.  Centralblatt  fiir  Bakteriologle,  Parasltenkunde  und 
Infektionskrankheiten,  Abteilung  2,  vol.  25,  No.  '5/9.  pp.  161-178.     Jena,  Nov.  30,  1909. 

2  Rogers,  L.  A.  Fermented  milks.  United  States  Department  of  Agriculture,  Bureau 
of  Animal  Industry,  Twenty-sixth  Annual  Report  (1909),  pp.  133-161.  Washington,  1911. 
Reprinted  as  Bureau  of  Animal  Industry  Circular  171,  Washington,  1911. 

'  Von  Freudenreich,  Edward,  and  ThSni,  J.  Sur  Taction  de  dlfif^rents  ferments  lac- 
tiques  sur  la  maturation  du  fromage.  Revue  G^n^rale  du  Lait,  vol  4,  No.  8,  pp.  169-181, 
Jan.  30;  No.  9,  pp.  200-209,  Feb.  15;  No.  10,  pp.  225-232,  Feb.  28;  No.  11,  pp.  247-259, 
Mar.  15.     Lierre,  1905. 

*  Bertrand,  Gabriel,  and  Weisweiller,  Gustav.  Action  du  ferment  bulgare  sur  le  lait. 
Annales  de  Institut  Pasteur,  vol.  20,  No.  12,  pp.  977-990.     Paris,  Dec.  25,  1906. 


42 


BACTEBIOLOGY   OF   CHEDDAR   CHEESE. 
Table  20. — Action  of  lactic  bacilli  in  milk. 


Cul- 
ture 

Acidity  pro- 
duced in  milk. 

Rotation  of  acid. 

Time 
of  cur- 
dling. 

Cul- 
ture 
No. 

Acidity  pro- 
duced in  milk. 

Rotation  of  acid. 

Time 
of  cur- 

No. 

10  days. 

45  days. 

10  days. 

45  days. 

dling. 

5 

Perct. 

0.324 

.643 

.68 

.69 

'"'.'89" 
.86 
.89 
.81 
1.02 
1.17 

Per  ct. 
0.91 
1.12 
1.05 
1.10 
1.26 
1.38 
1.21 
1.2 
1.36 

Days. 
11 

116.2 

92 
160 
165 
113 

71 

91 

85 
116.1 

96 

Per  ct. 
1.2 
1.24 
1.37 
1.41 
1.57 
1.6 
1.68 
1.73 
1.75 
1.89 

Per  ct. 
1.44 
1.4 
1.6 
1.57 
1.88 
1.75 

Daijf. 

152 

Inactive 

4 

62 

9 

Dextro 

4 

165.1 

4 

163 

9 

Dextro    . . 

4 

58A 

Inactive+levo .. 

4 

58B 

do 

4 

128 

1.89 
1.96 
2.31 

Dextro-f  inactive 

4 

116 

Inactive 

4 

120 

do 

3 

104 

1.46 

Dextro+ inactive 

4 

Heinemann  and  Hefferan^  report  the  formation  of  an  inactive 
acid,  while  White  and  Avery  ^  found  that  the  organisms  that  pro- 
duced a  large  amount  of  acid,  3  per  cent,  formed  an  inactive  acid, 
whereas  the  cultures  that  formed  acid  slowly  and  in  smaller  amounts 
showed  a  levo-rotary  power  at  the  end  of  24  hours,  but  in  older 
cultures  there  were  approximately  equal  amounts  of  levo  and  of 
inactive  acids. 

The  rotary  power  of  9  of  the  cultures  studied  was  determined  by 
Dr.  J.  M.  Currie.  Four  of  these  produced  inactive  acid ;  1  produced 
inactive  and  levo  acid,  with  a  predominance  of  the  inactive  form; 
2  produced  a  mixture  of  dextro  and  inactive  acids,  with  a  predomi- 
nance of  the  dextro-rotatory  form;  and  the  remaining  2  produced 
pure  dextro  acid.  One  culture  isolated  from  milk,  of  which  no  other 
study  was  made,  produced  pure  levo  acid.  With  11  cultures  isolated 
from  other  sources  than  milk  or  cheese,  only  the  pure  dextro  or  pure 
inactive  acids  were  found. 

This  type  of  organism,  then,  can  produce  inactive  acid,  or  active 
acid  of  either  modification,  or  a  mixture  of  the  inactive  acid  with 
either  of  the  active  acids.  All  of  these  forms  were  found  in  cultures 
isolated  from  Cheddar  cheese,  with  the  exception  of  the  pure  levo 
acid. 

TYPE  or  CURD  PRODUCED. 

In  most  of  the  more  active  cultures  the  reduction  of  litmus  and  the 
return  of  color  proceeded  in  exactly  the  same  manner  as  in  a  litmus- 
milk  culture  of  Bacterium  lactis  acidi.  On  the  other  hand,  curdling 
took  place  with  only  a  slight  or  partial  reduction  of  the  litmus  in 
some  cultures. 

*  Heinemann,  P.  O.,  and  Ilefferan,  Mary.  .\  study  of  Bacillus  Bulgarlcus.  Journal  of 
Infectious  Disoases,  vol   0,  No.  3,  pp.  ."04-318.     Chicago,  June  12,  1909. 

*  White,  Benjamin,  and  Avery,  Oswald  T.  Observations  on  certain  lactic  acid  bactoria 
of  the  so-called  Bulgarlcus  type.  Centralblatt  filr  Dakterlologle,  Parasltcnkunde  und 
Infektlonskrankheiten,  Abtellung  2,  vol.  25,  No.  5/9,  pp.  161-178.    Jena,  Nov.  30,  1909. 


DETAILED   STUDY   OP   LACTIC   BACILLI. 


43 


There  was  a  slight  gas  formation  in  most  of  the  cultures,  evidence 
of  which  was  found  in  the  slight  furrows  or  tiny  holes  which  some- 
times appeared  in  the  curd.  These  gas  holes  were,  however,  not 
always  found  in  curds  from  the  same  culture.  Although  most  of  the 
organisms  grew  in  lactose-agar  shake  cultures,  no  gas  formation  was 
noted  in  any  case. 

FERMENTATION  OF  SUGARS. 

Sixteen  of  the  cultures  were  tested  as  to  their  power  to  ferment 
sugars.  The  cultures  were  incubated  eight  days  at  37°  C.  The  test 
for  growth  and  fermentation  of  the  sugar  was  the  reddening  of 
litmus  paper.  The  results  are  given  in  Table  21.  All  of  the  strains 
tested  except  one.  No.  128,  grew  in  glucose  bouillon.  Many  of  the 
strains  made  no  growth  or  only  a  feeble  growth  (indicated  by  the 
sign  ± )  in  maltose  and  sucrose  bouillon,  although  all  except  No.  128 
grew  in  one  or  the  other,  or  both,  of  these  sugars. 

Table  21. — Growth  of  lactic  bacilli  in  culture  media. 


Cul- 
ture 
No. 

Growth  in  sugar 
bouillon. 

Growth 
on  lac- 
tose 
agar. 

Growth 
on  or- 
dinary 
gelatin. 

Cul- 
ture 
No. 

Growth  in  sugar 
bouillon. 

Growth 
on  lac- 
tose 
agar. 

Growth 
on  or- 
dinary 
gelatin. 

Glu- 
cose. 

Mal- 
tose. 

Su- 
crose. 

Glu- 
cose. 

Mal- 
tose. 

Su- 
crose. 

5 

152 
52 

165.1 

163 
58A 
58B 

a28 

116 

120 

104 

+ 

+ 
± 
+ 
+ 

+ 

+ 

+ 
± 

+ 
+ 

+ 
+ 

+ 

+ 

116.2 

92  . 
160 
165 
113 

71 

91 

85 
116.1 

96 

+ 

± 

+ 

+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 

+ 

+ 
+ 
+ 

+ 

+ 

+ 

-1- 
+ 
+ 
+ 
+ 
+ 
+ 

+ 

+ 
+ 
+ 
4- 

+ 

± 

+ 
+ 
± 
+ 
+ 
+ 

+ 
+ 

+ 
+ 
+ 

+ 

+ 

+ 

+ 

± 

+ 

+ 

±  indicates  feeble  growth. 


FORM    or   COLONIES. 


The  growth  upon  lactose  agar  and  ordinary  gelatin  was  deter- 
mined in  plate  cultures  on  the  same  media  that  was  used  for  the 
bacteriological  analysis  of  cheese.  Growth  was  obtained  in  lactose- 
agar  plate  cultures  from  every  strain  except  No.  128.  There  was  a 
good  growth  in  most  of  the  cultures,  although  with  some  only  a  few 
colonies  developed.  When  the  plates  were  incubated  for  several 
days  the  colonies  continued  to  increase  in  size,  sometimes  attaining 
a  diameter  of  1  mm.  The  surface  colonies  are  round  and  the  deep 
colonies  have  the  common  flattened  elliptical  form.  They  can  not 
be  distinguished  from  the  colonies  of  Bacterium  lactis  Midi.  This  is 
probably  one  reason  why  previous  investigators  have  failed  to  find 
this  type  of  organism  in  Cheddar  cheese. 

Growth  upon  plates  of  ordinary  gelatin  was  obtained  from  8  of 
the  cultures.     This,  however,  was  not  constant    for  some  strains 


44  BACTERIOLOGY   OF   CHEDDAR   CHEESE. 

which  developed  from  one  inoculation  failed  to  grow  from  another 
inoculation.  The  variable  growth  of  this  type  of  organism  in  plate 
cultures  can  account  for  some  of  the  inconsistent  results  obtained  in 
the  quantitative  analysis  of  cheese  by  means  of  lactose-agar  and  gela- 
tin plates. 

The  readiness  with  which  some  of  these  cultures  of  lactic  bacilli 
isolated  from  cheese  grow  upon  ordinary  media  is  rather  surprising, 
in  view  of  what  has  been  stated  by  many  authors  concerning  this 
property.  One  of  the  characteristics  generally  given  for  this  group 
of  organisms  is  their  meager  growth  upon  ordinary  media.  The  five 
cultures  studied  by  Von  Freudenreich  failed  to  grow  on  ordinary 
gelatin  or  on  gelatin  to  which  an  extract  of  cheese  had  been  added. 
Cohendy  ^  and  Lohnis  ^  found  these  organisms  hard  to  cultivate  be- 
cause of  their  reluctance  to  grow  upon  the  ordinary  nutrient  media. 
^Vhite  and  Avery  state  that  when  freshly  isolated  from  their  natural 
environment' — ^milk — they  do  not  develop  on  any  of  the  usual  nutrient 
media,  even  though  sugar  be  present.  Heineman  and  Heti'eran,  on 
the  other  hand,  state  that  they  grow  well  in  milk,  in  media  prepared 
from  milk,  or  if  glucose  is  added  to  the  ordinary  media.  All  of  the 
cultures  studied  were  isolated  by  means  of  lactose-agar  plates  with 
the  exception  of  No.  128,  which  was  obtained  in  pure  culture  in  tho 
dilution  cultures  in  milk. 

THERMAL  DEATH  POINT. 

The  resistance  of  the  cultures  to  heat  was  determined  on  3-day -old 
milk  cultures  which  had  been  diluted  and  shaken  with  water  and 
then  drawn  into  sterile  capillary  tubes.  The  tubes  were  held  for  30 
seconds  in  water  at  various  temperatures.  Most  of  the  cultures  were 
killed  at  some  temperature  between  62°  and  67°  C.  Two  cultures 
were  killed  between  60°  and  62.5°  C,  and  two  cultures  were  killed 
at  about  70°  C. 

MORPHOLOGY. 

The  bacilli  are  nonmotile  and  do  not  bear  spores.  In  a  single  mi- 
croscopic field  of  a  slide  prepared  from  a  pure  culture  the  organisms 
may  vary  in  length  from  2.5  microns  to  20  or  30  microns  or  more. 
Sometimes  the  filaments  are  very  long.  In  one  of  the  pure  cultures  a 
filament  was  found  75  microns  in  length.  In  one  of  the  mixed  cul- 
tures inoculated  from  cheese  there  was  found  a  filament  which  was 
about  960  microns  in  length,  extending  four  times  across  the  field  of 

1  Cohendy,  Michel.  Essals  d'accllmatJon  mlcroblenne  perslstante  dans  la  cavity  Intes- 
tinale.  Comptes  Rendus  Ilebdomadaires  des  Stances  de  la  Socl^tfi  de  Biologle,  vol.  60 
(ann^e  58,  tome  1),  No.  7,  pp.  364-366.     Paris,  Feb.  23,  1906. 

*L8hnl8,  F.  Versuch  einer  Grupplerung  der  Milchsaurebakterien.  Centralblatt  fflr 
Bakterlologie,  Parasltenkunde  und  Infektlonskrankbeiten,  Abteilung  2,  vol.  18,  No.  4/6, 
pp.  97-149.     Jena,  Mar.  14,  1907. 


DETAILED  STUDY   OP   LACTIC  BACILLI.  45 

the  microscope.  The  filaments  are  apparently  made  up  of  a  number 
of  individual  cells  which  for  some  reason  do  not  show  the  cell  divi- 
sions, for  frequently  chains  of  rods  are  found  instead  of  filaments. 
Occasionally  in  a  mixed,  culture  from  a  cheese  inoculation  filaments 
were  found  very  much  curled,  or  curled  at  one  end,  but  attempts  to 
isolate  a  culture  which  would  regularly  produce  curled  filaments 
alwaj's  resulted  in  securing  a  culture  with  the  usual  morphology.  No 
curled  filaments  were  ever  observed  in  pure  cultures.  The  width  of 
the  cells  in  a  pure  culture  seems  to  be  fairly  constant;  in  some  cultures 
the  individual  cells  are  all  slender,  in  other  cultures  they  are  all  com- 
paratively thick.  In  different  cultures  the  width  varies  from  0.4  to 
1.3  microns.    No  branching  forms  have  ever  been  observed. 

When  stained  with  methylene  blue  the  ends  of  the  cells  may  take 
the  stain  much  more  deeply  than  the  remainder  of  the  cell,  or  the 
deeply  stained  spots  may  be  scattered  irregularly  in  the  filament. 
Occasionally  the  deeply  stained  spots  appear  as  tiny  nodules  on  the 
rod.  These  characteristic  staining  properties  are  less  frequently  ex- 
hibited when  stained  by  the  Gram-Weigert  method.  In  a  weakened 
culture  filaments  are  sometimes  found  with  some  of  the  cells  Gram 
positive,  some  of  them  Gram  negative,  and  other  cells  taking  the  stain 
partially. 

Although  there  is  such  a  wide  difference  between  the  cultures  in 
regard  to  the  production  of  acidity  in  milk,  the  other  characteristics 
do  not  differentiate  a  culture  producing  a  low  acidity  from  one  which 
produces  a  high  acidity.  The  morphology,  however,  seems  to  bear 
some  relation  to  the  acidity  produced,  those  producing  a  high  acidity 
being  more  slender  than  those  producing  a  low  acidity. 

CONDITIONS  FOR  GROWTH  IN  CHEESE. 

It  has  been  pointed  out  that  in  their  development  in  cheese  the 
lactic  bacilli  follow  the  Bacterium  lactis  acidi  group,  and  that  the 
greater  part  of  the  growth  must  occur  after  the  disappearance  of  the 
sugar.  Since  lactic  acid  is  the  principal  by-product  of  the  fermenta- 
tion of  the  sugar,  it  might  seem  probable  that  the  lactic  bacilli  would 
make  use  of  this  as  a  source  of  carbon  and  of  energy.  Analyses  of 
cheese  show  no  decrease  in  lactic  acid  ^  at  the  period  of  development 
of  the  lactic  bacilli.  This  indicates  that  this  group  of  bacteria  does 
not  act  on  the  lactic  acid. 

The  action  of  the  pepsin  of  the  rennet  extract  activated  by  the 
lactic  acid  results  in  the  formation  of  peptones  from  the  paracasein. 
The  favorable  effect  of  peptone  on  the  lactic  bacilli  is  shown  in  the 

1  Suzuki,  S.  K. ;  Hastings,  E.  G. ;  and  Hart,  E  B.  The  production  of  volatile  fatty 
acids  and  esters  in  Cheddar  cheese  and  their  relation  to  the  development  of  flavor.  Wis- 
consin Agricultural  Experiment  Station,  Research  Bulletin  11.  Madison,  June,  1910. 
See  p.  135. 


46 


BACTERIOLOGY   OP   CHEDDAR   CHEESE. 


following  experiment.  Small  flasks  containing  100  c.  c.  of  sterile 
milk  were  inoculated  with  cultures  Nos.  5,  92,  91,  and  96.  To  one 
flask  of  each  set  there  had  been  added  before  sterilization  0.5  gram 
of  peptone,  and  to  another  flask  1  gram.  The  control  flask  received 
no  peptone.  In  Table  22  the  acidity  at  10  days  and  45  days  is  given. 
The  results  show  clearly  that  milk  to  which  peptone  has  been  added 
is  a  better  medium  than  plain  milk  for  the  growth  of  this  group  of 
organisms.  This  was  especially  true  in  the  case  of  culture  No.  5, 
which  usually  curdled  milk  in  about  11  days,  but  which  curdled  the 
milk  to  which  peptone  had  been  added  in  two  days — more  rapidly 
than  the  most  active  cultures  curdle  plain  milk.  The  time  of  curdling 
was  shortened  by  the  peptone  3  days  in  the  case  of  culture  No.  91, 
and  1  day  in  cultures  Nos.  92  and  96.  In  every  case  the  final 
acidity  at  42  days  was  considerably  greater  than  in  plain  milk,  and 
in  every  case  but  one  (culture  No.  91,  at  42  days)  there  was  a  greater 
development  of  acidity  in  the  milk  containing  1  gram  than  in  the 
milk  containing  0.5  gram  of  peptone. 

Incidentally,  this  experiment  shows  that  the  cessation  of  growth 
in  milk  wh3n  a  certain  percentage  of  acidity  is  reached,  which  is  a 
fairly  constant  percentage  for  each  particular  culture,  is  not  brought 
about  by  the  antiseptic  action  of  the  acid,  but  by  a  lack  of  suitable 
nitrogenous  food. 

Table  22. — Percentage  of  aciditp  produced  by  lactic  haciUi  in  milk  to  which 

peptone  has  been  added. 


Culture 
No, 

Acidity  in  10  days. 

Acidity  in  42  days. 

Plain 
milk. 

Milk  with 
0.5  gram 
peptone. 

Milk  with 

Igram 

peptone. 

Plain 
milk. 

Milk  with 
0.5  gram 
peptone. 

Milk  with 

1  gram 

peptone. 

5 
92 
91 
96 

Per  cent. 
0.324 
1.24 
l.CS 

1.58 

Per  cent. 
1.19 
1.4 
1.9 

1.87 

Per  cent. 
1.32 
1.58 
2.1 
1.96 

Percent.. 
0.855 
1.4 

Per  cent. 
1.35 
1.56 
2.42 
2.13 

Per  cent. 
2.34 

1.77 
2.29 
2.25 

1.93 

The  action  of  the  enzyms  present  in  the  cheese  curd  brings  about 
an  increasing  amount  of  soluble  nitrogenous  compounds  so  that  after 
one  month  of  ripening  about  18  per  cent  of  the  total  nitrogen  is  in 
the  foim  of  water-soluble  compounds.^  This  action  must  render  the 
curd  a  medium  favorable  to  the  growth  of  the  lactic  bacilli,  and 
particularly  to  those  strains  similar  to  culture  No.  5,  which  grow  so 
slowly  in  milk,  or  fail  to  grow  therein,  as  frequently  happens. 

The  dependence  of  this  group  of  organisms  upon  enzyms  from 
other  sources  is  shown  by  growing  them  together  with  other  types 

^  Van  Slyke,  L.  L.,  and  Hart,  E.  B.  Conditions  affecting  chemical  changes  in  cheese- 
ripening.  New  York  Agricultural  Experiment  Station,  Bulletin  23G.  Geneva,  July,  1903. 
See  p.  160. 


DETAILED  STUDY   OP   LACTIC   BACILLI. 


47 


of  bacteria.  Marshall  and  Farrand '  have  shown  that  when  Bac- 
terium lactis  acidi  grows  in  milk  together  with  certain  other  organ- 
isms there  is  an  associative  action  which  accelerates  the  production 
of  acid  and  the  proliferation  of  cells.  This  quickening  action  was 
found  to  occur  in  the  case  of  57  per  cent  of  the  cultures  grown  in 
association  with  Bacterium  lactis  acidi.  A  similar  action,  but  more 
pronounced,  takes  place  when  the  lactic  bacilli  grow  in  milk  together 
with  certain  other  types  of  organisms. 

Suspensions  in  water  were  made  of  a  48-hour  milk  culture  of  the 
lactic  bacillus  No.  104  and  also  of  48-hour  glucose  bouillon  cultures 
of  two  different  strains  of  liquefying  bacteria,  which  were  isolated 
from  poor  cheese.  This  particular  type  of  organism  has  not  been 
found  in  good  cheese.  Small  flasks  of  milk  were  inoculated  with 
various  dilutions  of  these  suspensions,  as  given  in  Table  23.  Since 
the  results  from  the  two  experiments  were  similar,  the  figures  for 
only  one  of  them  are  given. 


Tabi-f.  23. — Associative  action  of  lactic  bacilli  and  a  liquefying  organism. 


Flask 
No. 


I 

II 
III 
IV 
V 
VI 
VII 


Inoculation  with  liquefler. 


1  :1.000 

1  : 1,000 

1  : 1,000 

1:1,000 

1 :  100,000 

1  :  10,000,000... 
No  inoculation 


Inoculation  with  lactic  bacilli. 


No  inoculation 
1:10,000,000... 

1:100,000 

1:1,000 

1:1,000 

1:1,0C0 

1:1,000 


Acidity  |  Acidity      Acidity 
in  2  days,  in  3  days,  in  7  days. 


Per  cent. 

Per  cent. 

Per  cent. 

0.17 

0.17 

0.34 

.18 

.69 

1.44 

.26 

.85 

1.53 

.40 

.96 

1.44 

.36 

.91 

1.42 

.42 

.99 
.62 

.31 

1.36 

It  will  be  seen  that  in  2  days  the  production  of  acidity  was  accel- 
erated in  the  cultures  which  received  an  inoculation  of  the  liquefier. 
This  was  more  pronounced  in  3  days,  but  less  pronounced  in  7  days, 
when  the  flask  which  received  no  inoculation  of  the  liquefier  showed 
only  a  small  amount  of  acidity  less  than  the  flasks  inoculated  with 
both  cultures. 

Other  experiments  were  made  with  cultures  of  coccus  forms  iso- 
lated from  cheese.  The  liquefying  action  of  these  was  not  pro- 
nounced, yet  they  exerted  a  stimulating  effect  on  the  lactic  bacilli. 
Reference  to  the  coccus  forms  found  in  cheese  will  be  made  later. 


SOLVENT  EFFECT  ON  MILK  PROTEINS. 

Von  Freudenreich  was  the  first  to  demonstrate  the  fact  that  lactic 
bacilli  exert  a  digestive  effect.     The  same  action^  was  shown  for  cul- 

1  Marshall,  Charles  E.,  and  Farrand,  Bell.  Bacterial  associations  in  the  souring  of 
milk.  Michigan  Agricultural  Experiment  Station,  Special  Bulletin  42.  East  Lansing, 
Mar.,  1908. 

-  Hastings,  E.  G.,  Hammer,  B.  W.,  and  Hoffman,  C.  Studies  on  the  bacterial  and 
leucocyte  content  of  milk.  Wisconsin  Agricultural  Experiment  Station,  Research  Bulletin 
6.     Madison,  June,  1909.     See  p.  202. 


48 


BACTERIOLOGY  OP  CHEDDAR   CHEESE. 


tures  isolated  from  milk  and  has  also  been  demonstrated  by  more 
recent  investigators. 

The  solvent  effect  of  8  of  the  cultures  studied  has  been  determined 
by  the  anah'ses  of  milk  cultures  after  3  months'  incubation.  .The 
results  are  given  in  Table  24. 

Table  24. — The  solvent  effect  of  lactic  hacilli  on  millc  proteins. 


Culture. 

Soluble  N. 

inlOOc.c. 

of  milk. 

Increase  in  soluble  N 
inlOOc.c.  of  milk. 

Sterile  milk 

Oram, 
0.064 
.072 
.076 
.080 
.081 
.090 
.092 
.096 
.104 

Oram. 

o.'oos 

.012 
.016 
.017 
.026 
.028 
.032 
.040 

Percent 

12.' 5 
18.7 
25.0 
.     26.5 
40.6 
43.7 
50.0 
62.5 

Ii6 

113 

152  

120 

116.1 

116.2 

71 

52 

COCCUS  FORMS  IN  CHEDDAR  CHEESE. 

Several  investigators  have  mentioned  coccus  forms  in  relation  to 
cheese  ripening.  Von  Freudenreich  and  Thoni  ^  found  regularly  a 
liquefying  micrococcus  in  quite  large  numbers  in  fresh  Emmental 
cheese.  They  made  a  number  of  experimental  cheeses,  using  this 
type  of  organism  alone  as  a  starter  or  together  with  lactic  starters. 
They  concluded  that  when  present  in  too  great  numbers  the  liquefy- 
ing micrococci  produced  bitterness,  but  that  they  disappeared  quite 
rapidly  in  practical  cheesemaking.  Gorini  ^  isolated  a  coccus  with 
the  property  of  peptonizing  casein  in  an  acid  medium  from  Grana, 
Emmental,  and  Edam  cheese.  He  found  these  forms  not  only  in  the 
fresh  cheese,  but  in  cheese  several  months  old.  He  thought  that 
they  we're  not  all  of  the  same  type,  and  he  related  them  to  the  normal 
flora  of  the  udder.  In  a  more  recent  publication  ^  Gorini  states  that 
later  investigations  have  confirmed  his  opinion  that  acid-producing 
peptonizing  ferments  are  important  in  the  ripening  of  cooked  cheeses 
and  that  the  fundamental  flora  of  Grana  and  other  cooked  cheeses  is 
composed  of  two  types  of  bacteria;  (1)  lactic  acid  bacteria,  and  (2) 
acid-producing,  peptonizing  ferments.  In  the  latter  class  he  in- 
cludes bacillus  forms  with  those  properties  and  divers  types  of  cocci. 

No  reference  to  coccus  forms  having  been  found  in  considerable 

*  Von  Freudenreich,  Edward,  and  Th5n!,  J.  Sur  les  bact^rles  du  lalt  normal  et  leurs 
rapports  avec  la  maturation  des  fromages.  Revue  G6n6rale  du  Lait,  vol.  2,  No.  11,  pp. 
241-247,  Mar.  15  ;  No.  12,  pp.  271-280,  Mar.  30,  Llerre,  1903. 

'Gorini,  C.  Sur  la  presence  de  bact^ries  productrices  d'acidit6  et  de  pr^sure  dans  le.s 
fromages  en  maturation.  Rue  G4n6rale  du  Lait,  vol.  3,  No.  22,  pp.  505-510.  Lierre, 
Aug.  31,  1004. 

'  Gorini,  C.  Studi  sulla  fabbrlcazione  del  fromaggi  Grana,  ecc.  Italy-MInistero  dl 
Agricoltura,  Industrlae  Commercla.  Bollettino.  Anno  9,  vol.  1,  ser.  C,  No.  6,  pp.  9-17. 
Rome,  .luno,  1910. 


coccus  FORMS  IN   CHEDDAR  CPIEESE.  49 

numbers  has  been  made  in  the  literature  on  Cheddar  cheese.  Hard- 
ing and  Prucha  ^  report  the  presence  of  acid-producing,  liquefying 
coccus  forms  in  9  out  of  10  cheeses  studied.  They  state  that  these 
forms  occurred  sufficiently  often  to  suggest  that  they-  might  play 
some  part  in  the  ripening  changes,  but  that  they  made  little  headway 
in  the  cheese,  and  their  number,  as  compared  to  the  total  germ  con- 
tent of  the  cheese,  was  relatively  insignificant. 

Coccus  forms  which  produce  a  small  amount  of  acidity  were  oc- 
casionally found  to  be  the  predominating  type  of  organism  in  the 
cheeses  studied,  as  shown  by  the  milk  cultures  from  high  dilutions 
of  the  cheese.  Unfortunately,  with  the  dilution  method  of  analysis 
this  type  can  be  differentiated  only  when  it  predominates,  for  if  it 
occurs  in  a  culture  with  the  other  cheese  organisms  it  can  not  be 
distinguished  with  certainty  from  the  Bacterium  lacfis  acidi  type  in 
a  microscopic  preparation,  and  the  titration  of  the  milk  gives  no 
information,  since  its  production  of  acidity  is  less  than  either  the 
lactic  bacilli  or  Bacterium  lactis  acidi.  However,  this  type  has  been 
found  to  predominate  at  some  time  or  other  in  11  of  the  13  cheeses 
examined  by  this  method. 

In  4  of  the  11  cheeses  the  maximum  number  of  coccus  forms  found 
tvas  100^000,000  per  gram,  in  4  other  cheeses  1,000,000,000,  and  in  the 
3  remaining  10,000,000,000  per  gram.  The  time  at  which  they  pre- 
dominated varied  from  the  14th  to  the  161st  day.  This  would  indi- 
cate that  they  increase  early  in  the  ripening  period  and  maintain 
such  numbers  for  a  considerable  period.  The  coccus  forms  have 
more  or  less  of  a  liquefying  action  on  gelatin ;  a  few  show  a  decided 
action,  some  none  at  all,  while  the  majority  produce  a  minute  de- 
pression in  the  gelatin  around  the  colony.  Their  action  is  not  com- 
parable with  that  of  those  organisms  usually  classed  as  liquefying 
bacteria.  They  produce  small  crystals  in  milk  that  enables  one  to 
differentiate  them  from  Bacterium  lactis  acidi.  The  nature  of  these 
crystals  has  not  yet  been  determined. 

The  various  cultures  produced  acidities  in  milk  varying  from 
0.35  to  0.80  per  cent. 

As  has  been  previously  mentioned,  all  of  the  colonies  from  a  cir- 
cumscribed area  on  the  lactose-agar  plates  prepared  from  the  cheeses 
last  examined  were  inoculated  into  milk.  The  formation  of  the 
crystals  was  used  to  distinguish  the  coccus  forms.  In  some  cheeses, 
especially  those  with  a  low  germ  content,  the  cocci  have  made  up 
from  10  to  40  per  cent  of  the  bacteria  as  determined  by  lactose-agar 
plate  cultures.  In  other  cheeses  they  have  not  been  detected  in  many 
of  the  examinations  made,  and  when  found  were  in  minor  numbers. 

1  Harding,  H.  A.,  and  Prucha,  M.  J.  The  bacterial  flora  of  Cheddar  cheese.  New  York 
Agricultural  Experiment  Station,  Technical  Bulletin  8.     Geneva,  Dec,  1908.     See  p.  184. 


60  BACTERIOLOGY   OF   CHEDDAR   CHEESE. 

It  should  be  mentioned,  however,  that  this  method  of  detecting 
the  various  kinds  of  organisms  in  cheese  is  subject  to  considerable 
trror,  especially  when  certain  forms  are  present  in  much  smaller 
numbers  than  other  forms,  as  is  the  case  with  the  coccus  forms  as 
compared  with  the  lactic  bacteria  of  both  groups  in  many  of  the 
cheeses  examined.  The  error  can  be  reduced  by  making  subcultures 
of  a  greater  number  of  colonies.  By  the  extension  of  this  method 
or  by  the  use  of  some  differentiating  medium  they  may  be  shown  to 
make  up  a  considerable  part  of  the  flora  of  normal  Cheddar  cheese. 

CHROMOGENIC    COCCI. 

The  chromogenic  cocci  can  be  distinguished  on  the  plate  cultures 
when  they  are  present  in  appreciable  numbers.  In  a  portion  of  the 
work  the  number  of  colonies  of  chromogens  appearing  on  gelatin 
plates  was  determined.  The  results  seemed  to  indicate  a  relative 
increase  late  in  the  ripening  period.  As  has  previously  been  pointed 
out,  it  is  impossible  to  determine,  in  the  case  of  any  group  of  organ- 
isms for  which  no  means  of  differentiation  from  other  groups  has 
been  foundj  whether  growth  is  taking  place  in  cheese  or  not. 

It  is  characteristic  of  the  chromogenic  cocci  that  they  persist  and 
possibly  grow  under  conditions  which  rapidly  destroy  less  resistant 
types.  They  are  present  in  butter,  in  which  food  material  is  limited, 
and  in  which  the  moisture  represents  a  saturated  solution  of  sodium 
chlorid.  Efforts  were  made  to  determine  their  number  in  cheese  by 
plating  on  gelatin  containing  3  or  4  per  cent  of  sodium  chlorid.  The 
results  were  quite  satisfactory  as  far  as  the  chromogenic  types  were 
concerned,  but  since  many  other  coccus  forms  did  not  grow  thereon 
its  use  was  given  up. 

In  some  cheeses  very  few  chromogenic  forms  have  been  found, 
while  in  others  of  similar  quality  they  have  been  present  quite  con- 
sistently in  considerable  numbers.  In  cheese  which  contained  more 
than  the  usual  amount  of  salt  they  made  up  a  relatively  greater  pro- 
portion of  the  flora  than  in  other  cheese. 

LIQUEFYING  ORGANISMS. 

Attention  has  been  directed  to  those  organisms  that  show  a  pro- 
nounced liquefying  action  on  gelatin  and  casein.  The  results  have 
been  confirmatory  of  a  statement  made  earlier  that  they  can  not  be 
considered  of  importance  in  the  ripening  of  Cheddar  cheese  since 
they  are  not  consistently  present  in  sufficient  numbers  to  exert  any 
effect. 


SEQUENCE  IN  DEVELOPMENT  OF  BACTERIAL  GROUPS.  51 

THE     SEQUENCE    IN    DEVELOPMENT     OF    BACTERIAL     GROUPS    IN 

CHEDDAR  CHEESE. 

In  the  first  part  of  this  bulletin  it  was  stated  that  the  normal 
ripening  of  each  kind  of  cheeese  is  to  be  looked  upon  as  a  problem 
in  the  ecology  of  microorganisms,  and  that  the  only  group  of  organ- 
isms which  has  been  found  by  previous  investigators  in  every 
Cheddar  cheese  in  great  numbers  is  the  Bdcterium  lactis  acidi  group. 
The  work  herein  reported  proves  that  another  group  of  bacteria,  the 
so-called  B.  hulgancus  group,  develops  after  the  first,  and  that  it 
reaches  approximately  equal  numbers. 

Each  of  these  groups  of  organisms  produces  certain  changes  in  the 
cheese  mass,  consuming  the  same  class  of  substances  and  giving  out 
the  same  by-products  in  every  cheese  which  ripens  normally.  The 
second  group  takes  up  the  work  of  decomposition  at  a  certain  stage 
of  the  ripening,  and  brings  about  its  own  peculiar  changes  which 
prepare  the  cheese  mass  for  possibly  still  other  types  of  organisms, 
and  so  on  to  the  end  of  the  ripening. 

If  a  certain  sequence  of  groups  of  microorganisms  is  essential  for 
the  preparation  of  a  certain  product  from  raw  material,  and  if  the 
various  members  of  the  sequence  find  favorable  conditions  for  growth 
in  the  raw  material,  the  resulting  product  will  depend  on  the  first 
member  of  the  normal  sequence  developing  to  the  necessary  degree 
before  the  second  appears.  If  the  first  is  overwhelmed  by  the  addi- 
tion of  great  numbers  of  the  second,  the  decomposition  changes  will 
not  be  normal.  The  presence  of  great  numbers  of  organisms  of  the 
first  member  of  the  sequence  will  not  cause  a  disturbance  in  the 
normal  decomposition  changes.  Thus,  in  the  manufacture  of  Ched- 
dar cheese,  cultures  of  Bacteriimi  lactis  acidi  are  added. 

The  other  essential  groups  of  microorganisms  are  present  in  the 
milk  in  small  numbers,  but  as  conditions  become  favorable  they  de- 
velop in  the  cheese,  and  a  typical  Cheddar  cheese  results. 

If  heavy  inoculations  of  lactic  bacilli  are  made  in  milk  which  con- 
tains a  small  number  of  Bacterium  lactis  acidi  the  normal  ecological 
balance  will  be  destroyed,  and  the  result  will  not  be  a  normal  product. 
Experiments  have  been  made  along  this  line  by  the  use  of  milk  which 
has  been  pasteurized  in  such  a  manner  that  the  result  has  been  the 
great  reduction  of  all  groups  of  bacteria  therein,  but  the  total  destruc- 
tion of  none.  In  its  decomposition  such  milk  is  similar  to  raw  milk, 
which  contains  but  few  bacteria.  If  cheese  is  prepared  from  such 
milk  after  the  addition  of  a  culture  of  Bacterium  lactis  acidi,  it  may 
ripen  in  a  quite  normal  manner.  The  development  of  the  flavor  is 
usually  slower  than  in  a  cheese  made  from  raw  milk.  If  to  this  same 
milk  is  added  a  culture  of  lactic  bacilli  instead  of  Bacterium  lactis 
acidi,  the  ripening  is  not  nearly  so  normal.  The  result  is  an  increased 
rate  of  ripening  and  the  production  of  an  abnormal  flavor. 


52  BACTERIOLOGY   OF   CHEDDAR   CHEESE. 

This  work  also  indicates  that  it  is  often  useless  to  attempt  to 
establish  the  role  of  any  organism  in  cheese  ripening  by  the  addition 
of  cultures  to  the  milk  to  be  used,  since  thereby  the  natural  equi- 
librium is  destroyed,  and  the  results  obtained  indicate  that  the  addi- 
tion has  injured  the  product,  and  hence  the  conclusion  is  drawn  that 
the  organism  added  is  not  only  not  essential,  but  even  harmful, 
although  the  organism  may  be  an  essential  factor  in  the  decomposi- 
tion changes  when  developing  in  its  normal  sequence. 

The  constant  presence  in  large  numbers  is  the  only  certain  proof 
of  the  importance  of  an  organism  or  group  of  organisms  in  the 
ripening  of  any  cheese. 

SUMMARY. 

1.  From  the  same  raw  materials  various  kinds  of  cheese  are  pre- 
pared, which  differ  especially  in  flavor.  The  factors  that  determine 
whether  a  cheese  to  be  prepared  from  a  given  mass  of  milk,  rennet, 
and  salt  is  to  be  of  one  kind  or  another  are  to  be  found  in  the  methods 
of  the  cheese  maker,  who  is  able  to  vary  in  one  way  or  another  the 
composition  of  the  cheese,  with  the  result  that  conditions  are  estab- 
lished that  favor  or  retard  the  growth  of  the  groups  of  micro- 
organisms, which  must  be  the  determining  factors  between  different 
kinds  of  cheese. 

2.  The  only  group  of  bacteria  found  constantly  in  great  numbers 
in  Cheddar  cheese  by  previous  investigators  is  the  Bacterium  laetis 
acidi  group.  The  functions  of  this  group  in  Cheddar  cheese  are, 
through  their  chief  by-product,  lactic  acid — 

{a)  To  favor  the  curdling  of  milk  by  rennet. 

{h)  The  bacteria  of  the  milk  are  held  in  great  part  in  the  curd. 
Through  the  acid  they  influence  the  shrinking  of  the  curd  and  expul- 
sion of  the  whey. 

(c)  The  acid  so  changes  the  nature  of  the  curd  as  to  cause  "  mat- 
ting." 

{d)  The  acid  activates  the  pepsin  of  the  rennet  extract. 

{e)  The  acid  prevents  the  growth  of  putrefactive  bacteria  in  the 
cheese. 

3.  It  has  been  shown  that  Bacterium  laetis  acidi  is  able  to  form 
acid  in  the  absence  of  the  living  cell. 

4.  The  development  of  Bacterium  laetis  acidi  is  followed  by  the 
growth  of  another  group  of  acid-forming  bacteria,  the  Bacillus  Bul- 
garicus  group.  They  reach  numbers  comparable  with  those  of  the 
first  group,  reaching  their  maximum  numbers  within  the  first  month 
of  the  ripening.  Since  they  develop  after  the  fermentation  of  the 
sugar,  they  must  have  some  other  source  of  carbon  and  of  energy 
than  milk  sugar. 

5.  It  is  probable  that  coccus  forms  are  constantly  found  in  large 
numbers  in  Cheddar  chc6se. 

o 


,^  LIBRAHY  FACILITV 


001137  153     1 


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